Qsox1 as an anti-neoplastic drug target

ABSTRACT

The present invention provides methods for tumor treatment by administering an inhibitor of quiescin sulfhydryl oxidase 1 (QSOX1), compositions comprising such inhibitors, and methods for identifying such inhibitors.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/169,612 filed on Jan. 31, 2014, which is a continuation-in-part (CIP)of U.S. patent application Ser. No. 13/847,930 filed on Mar. 20, 2013,now U.S. Pat. No. 8,946,186, which claims the benefit of priority under35 USC §119(e) of U.S. Provisional Patent Application No. 61/722,396filed on Nov. 5, 2012, and is a continuation-in-part (CIP) of PCT PatentApplication No. PCT/US2011/052122 filed on Sep. 19, 2011, which claimsthe benefit of priority under 35 USC §119(e) of U.S. Provisional PatentApplication No. 61/384,502 filed on Sep. 20, 2010. The contents of theabove applications are all incorporated by reference as if fully setforth herein in their entirety.

BACKGROUND

Pancreatic ductal adenocarcinoma (PDA) is a disease that carries a poorprognosis. It is often detected in stage III resulting in anunresectable tumor at the time of diagnosis. However, even if pancreaticcancer is surgically resected in stage I or II, it may recur at ametastatic site (1, 2). Currently, patients diagnosed with pancreaticductal adenocarcinoma have less than a 5% chance of surviving past fiveyears (3).

Breast adenocarcinoma is the most common cancer diagnosed in womenthroughout the world. In 2012, an estimated 226,870 new cases ofinvasive breast cancer are expected to occur among US women, and anestimated 39,510 breast cancer deaths.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides methods for tumortreatment, comprising administering to a subject having a tumor anamount effective of an inhibitor of quiescin sulfhydryl oxidase 1(QSOX1) expression and/or activity, or a pharmaceutically acceptablesalt thereof, to treat the tumor. In one embodiment, the inhibitor ofQSOX1 is selected from the group consisting of anti-QSOX1 antibodies,QSOX1-binding aptamers, QSOX1 antisense oligonucleotides, QSOX1 siRNA,and QSOX1 shRNA. In another embodiment, the tumor is a tumor thatover-expresses QSOX1 compared to control. In a further embodiment, thesubject is one from which tumor-derived QSOX1 peptides can be obtained.In a further embodiment, the tumor is a pancreatic tumor, and preferablya pancreatic adenocarcinoma. In a still further embodiment, the methodis for limiting tumor metastasis.

In a second aspect, the present invention provides isolated nucleicacids, comprising or consisting of antisense, siRNA, miRNA, and/or shRNAmolecules having a nucleic acid sequence that is perfectly complementaryat least 10 contiguous nucleotides of QSOX1 as shown in SEQ ID NO:1 andSEQ ID NO:2 or RNA equivalents thereof; and/or fragments of the nucleicacid molecule. In a preferred embodiment, the isolated nucleic acidscomprising sequences from the group consisting of

(SEQ ID NO: 11) 5′-A(T/U)C(T/U)ACA(T/U)GGC(T/U)GACC(T/U)GGAA-3′ (SEQ IDNO: 12) 5′-AGGAAAGAGGG(T/U)GCCG(T/U)(T/U)C(T/U)(T/U)-3′, (SEQ ID NO: 13)5′-GCCAA(T/U)G(T/U)GG(T/U)GAGAAAG(T/U)(T/U) (T/U)-3′, (SEQ ID NO: 14)5′-GCCAAGAAGG(T/U)GAAC(T/U)GGA(T/U)(T/U)-3′. (SEQ ID NO: 15)5′-CCGGACAA(T/U)GAAGAAGCC(T/U)(T/U)(T/U)-3′ (SEQ ID NO: 16)5′-(T/U)C(T/U)AGCCACAACAGGG(T/U)CAA(T/U)-3′ (SEQ ID NO: 17)5′-ATCTACATGGCTGACCTGGAA-3′, (SEQ ID NO: 18)5′-AGGAAAGAGGGTGCCGTTCTT-3′, (SEQ ID NO: 19)5′-GCCAATGTGGTGAGAAAGTTT-3′, (SEQ ID NO: 20)5′-GCCAAGAAGGTGAACTGGATT-3′, (SEQ ID NO: 21)5′-CCGGACAATGAAGAAGCCTTT-3′; (SEQ ID NO: 22)5′-TCTAGCCACAACAGGGTCAAT-3′; and (SEQ ID NO: 26)5′-CCGGGCCAATGTGGTGAGAAAGTTTCTCGAGAAACTTT CTCACCACATTGGCTTTTTG-3′.

In one embodiment of this second aspect of the invention, the isolatednucleic acids present in a short hairpin RNA (shRNA). In a furtherembodiment, the shRNA is of the general formula:

(SEQ ID NO: 23) CCGG-X1-CTCGAGAAACTTTCTCACCACATTGGCTTTTTG-3′wherein X1 is a nucleic acid sequence selected from the group consistingof

(SEQ ID NO: 11) 5′-A(T/U)C(T/U)ACA(T/U)GGC(T/U)GACC(T/U)GGAA-3′ (SEQ IDNO: 12) 5′-AGGAAAGAGGG(T/U)GCCG(T/U)(T/U)C(T/U)(T/U)-3′, (SEQ ID NO: 13)5′-GCCAA(T/U)G(T/U)GG(T/U)GAGAAAG(T/U)(T/U)(T/U)- 3′, (SEQ ID NO: 14)5′-GCCAAGAAGG(T/U)GAAC(T/U)GGA(T/U)(T/U)-3′. (SEQ ID NO: 15)5′-CCGGACAA(T/U)GAAGAAGCC(T/U)(T/U)(T/U)-3′ (SEQ ID NO: 16)5′-(T/U)C(T/U)AGCCACAACAGGG(T/U)CAA(T/U)-3′ (SEQ ID NO: 17)5′-ATCTACATGGCTGACCTGGAA-3′, (SEQ ID NO: 18)5′-AGGAAAGAGGGTGCCGTTCTT-3′, (SEQ ID NO: 19)5′-GCCAATGTGGTGAGAAAGTTT-3′, (SEQ ID NO: 20)5′-GCCAAGAAGGTGAACTGGATT-3′, (SEQ ID NO: 21)5′-CCGGACAATGAAGAAGCCTTT-3′; and (SEQ ID NO: 22)5′-TCTAGCCACAACAGGGTCAAT-3′.

In a third aspect, the present invention provides recombinant expressionvectors comprising the isolated nucleic acid of any embodiment of thesecond aspect of the invention operatively linked to a promoter.

In a fourth aspect, the present invention provides recombinant hostcells comprising the recombinant expression vector of the third aspectof the invention.

In a fifth aspect, the present invention provides pharmaceuticalcompositions, comprising

(a) the isolated nucleic acid of any embodiment of the second aspect ofthe invention, the recombinant expression vector of any embodiment ofthe third aspect of the invention, or the recombinant host cell of anyembodiment of the fourth aspect of the invention; and

(b) a pharmaceutically acceptable carrier.

In a sixth aspect, the present invention provides methods foridentifying candidate compounds for treating a tumor, comprising

(a) contacting tumor cells capable of expressing QSOX1 with one or morecandidate compounds under conditions suitable for expression of QSOX1;

(b) determining a level of QSOX1 expression and/or activity in the tumorcells and comparing to control;

wherein a compound that decreases QSOX1 expression and/or activity inthe tumor cells relative to control is a candidate compound for treatinga tumor.

In one embodiment, the tumor cells are pancreatic tumor cells,preferably pancreatic adenocarcinoma cells.

In another aspect, the invention provides methods for prognosing atumor, comprising

(a) determining a QSOX1 expression level in a sample from a subjecthaving a tumor;

(b) comparing the QSOX1 expression level to control; and

(c) prognosing the progression of the tumor in the subject.

DESCRIPTION OF THE FIGURES

FIG. 1: QSOX1 is highly expressed in tumor cell lines but is notexpressed in adjacent normal cells. Previously, our lab discovered ashort peptide, NEQEQPLGQWHLS (SEQ ID NO:3), in patient plasma throughLC-MS/MS. We were able to map this short, secreted peptide back to anunderstudied parent protein, QSOX1-L. A.) Diagram showing the two splicevariants of QSOX1, QSOX1-Short (S) and -Long (L), both contains athioredoxin 1(Trx1) and ERV/ALR functional domains as well as structuralthioredoxin 2 (Trx2) and helix rich region (HRR). QSOX1-L contains apredicted transmembrane (TM) domain. The peptide NEQEQPLGQWHLS (SEQ IDNO:3), maps back to QSOX1-L, and found to be secreted in pancreaticcancer patients but not in normal samples. The commercially availableantibody recognizes the first 329 amino acids of both QSOX1-S and -L.B.) Immunohistochemistry of normal (left) and tumor (right) pancreatictissue sections that have been stained with the anti-QSOX1 showing tumorspecific staining in pancreatic ducts but not in adjacent non-tumorcells. C.) Western blot analysis of patient tumor as well as adjacentnormal tissue indicates that QSOX1-S is the dominant splice variantexpressed. D.) Western blot showing QSOX1 expression in transformednormal pancreatic cells (HPDE6) and Human Pancreatic Adenocarinoma Cells(Panc-1, CFPac-1, BxPC3, and Capan1) shows that our in vitro systemmimics that of the in vivo QSOX1 expression as shown above using IHC.

FIG. 2: Reduced expression of QSOX1 in BxPC3 and Panc-1 cells leads to asignificant decrease in cell growth. To determine the phenotypepresented due to the expression of QSOX1 in tumor cells we employedshRNA specific to QSOX1 to reduce the expression of QSOX1 in A.) BxPC3(Percent Decrease in sh742—56%; sh528—40%; sh616—28%) and B.) Panc-1(Percent Decrease in sh742—64%; sh528—46%; sh616—18%) cells and furtherevaluated cell growth, cell cycle, apoptosis, and invasion/metastasis.C.) MTT assay on shRNA treated BxPC3 and Panc-1 cells assayed on day 1,2, and 5. Data represents averages±standard deviation. Significance *,P<0.05; **, P<0.01.

FIG. 3: Reduced expression of QSOX1 in BxPC3 and Panc-1 cells leads toan increase in annexin V/propidium iodide positive cells. A.) ApoptosisAnalysis (Annexin V/Propidium Iodide) was performed on BxPC3 and Panc-1cells in which QSOX1 was reduced using shRNA. Plots show representativedata from one of three individual experiments for gated samples ofUntreated, Scramble, sh742, sh528 and sh616. The percentages representthe number of cells that are annexin V positive (Lower Right), annexinV/propidium iodide double positive (Upper Left), or propidium iodidepositive (Upper Right). Data was calculated using Cell Quest Prosoftware.

FIG. 4: Reduced expression of QSOX1 in BxPC3 and Panc-1 cells leads to asignificant decrease in cellular invasion. A.) Untreated BxPC3 and B.)Untreated Panc-1 cells were treated with Scramble, sh742, sh528 andsh616 shRNA's specific for QSOX1 and seeded in the top chamber ofMatrigel™ invasion wells and allowed to incubate for 18 hours.Representative 10×, 20×, and 40× images are presented. In the BxPC3sh742, sh528 and sh616 treated cells there was an 84%, 84%, and 79%decrease in cells that were able to break down the basement membranecomponents of the Matrigel™ and invade to the underside of the membrane,respectively. While in Panc-1 sh742, sh528 and sh616 cells there was a76%, 76%, and 63% decrease in cells that were able to degrade theMatrigel™ and invade through the membrane. Graphs representaverage±standard deviation (BxPC3 n=6; PANC-1 n=3), significance *,P<0.05, **, P<0.005.

FIG. 5: Reduced expression of QSOX1 leads to a decrease in secretedproMMP-9 in BxPC3 and proMMP-2 Panc-1 cells. Gelatin zymography of A.)BxPC3 and B.) Panc-1 conditioned media showing a decrease in MMP-9homodimers (MMP-9 Complex) (240 and 130 kDa), pro-MMP9 (92 kDa),pro-MMP2 (72 kDa) and active MMP-2 (a-MMP2, 66 kDa). Using Image J wewere able to quantify the percent decrease in proMMP-9 expression inBxPC3 (Decrease in QSOX1, sh742—65%; sh528—47%; sh616—10%) and Panc-1proMMP-2 (Decrease in QSOX1, sh742—70%; sh528—56%; sh616—15%). C.)Western blot analysis of MMP-2 and -9 on conditioned serum free mediafrom shRNA treated BxPC3 and Panc-1 cells. D.) The effect of shRNAmediated knockdown of QSOX1 on the expression of QSOX1-S, QSOX1-L,MMP-2, and MMP-9 in BxPC3 and Panc-1 shRNA treated cells was analyzed byquantitative real time PCR analysis. The graph represents relative geneexpression calculated as ΔΔCq using GAPDH as the endogenous referencegene.

FIG. 6. Cellular proliferation was measured by MTT assay 4 dayspost-transfection shows that knockdown of QSOX1 short and long formexpression by transient lipid-mediated transfection of shRNA results ina decrease in tumor cell viability as measured by a cellularmitochondrial respiration assay (MTT assay). This supports the idea thatshRNA or other RNAi species could be used as a drug to suppress QSOX1protein expression, and inhibit the growth, invasion, and metastases oftumors over-expressing QSOX1. No shRNA—Untreated BxPC3 Cells; ScrambledshRNA—Negative control. BxPC3 cells transfected with scrambled shRNAcontrol vector; shRNA1—BxPC3 cells transfected with shRNA1 for 4 days;shRNA2—BxPC3 cells transfected with shRNA2 for 4 days.

FIG. 7. Invasion Assay shows that by knocking down QSOX1 with the aboveshRNA we are able to see a decrease in tumor cell invasion. Cells weretransfected, allowed to recover for 4 days, resuspended in serum freemedia and added to 8 um (pore size) microwell inserts coated withMatrigel™. The inserts were placed in cell culture media containing 10%fetal bovine serum such that the bottom half of the outside of theinsert was exposed to the media. After 24 hours incubation at 37° C., 5%CO₂, the inserts were removed and washed in buffer. Cells that were ableto degrade the Matrigel™ coating and invade through the 8 um pores inthe insert were counted on the underside of the well. Using theMatrigel™ assay we are able to show that when we knock down QSOX1 inBxPC3 (pancreatic cancer cells) the cells are no longer able to degradethe basement membrane components resulting in a decrease in cellularinvasion. Untreated—Untreated BxPC3 Cells; Scrambled—BxPC3 cellstransfected with scrambled shRNA control vector. Invasion measured at 4days post-transfection; shRNA1—BxPC3 cells transfected with shRNA1 andinvasion measured 4 days post-transfection; shRNA2—BxPC3 cellstransfected with shRNA2 and invasion measured 4 days post-transfection;shRNA1/2—BxPC3 cells transfected with a combined shRNA1 and shRNA2.Cells were measured for invasion properties 4 days post-transfection.

FIG. 8 is a graph showing MIAPaCa3 pancreatic tumor cell xenograftgrowth rate as a result of shRNA knockdown assays described in Example3. Human pancreatic tumor cells (MIAPaCa2) were transduced with alentivirus encoding shQSOX1 (sh528 and sh742) and shScramble (control).One million MIAPaCa2 cells were mixed with Matrigel™ and used toinoculate nude mice (5 mice/group) on day 0. After day 12, tumor growthwas measured every 3 days (x-axis) and reported as “Tumor volume” on theY-axis. “Untreated” indicates that tumor cells were not transduced.

FIG. 9 is a graph showing MIAPaCa3 pancreatic tumor cell xenograft finaltumor weights as a result of shRNA knockdown assays described in Example3.

FIG. 10 QSOX1 promotes tumor cell invasion. a) MCF7, b) BT549 and c)BT474 cells transduced with shScramble, sh742 and sh528 shRNAs wereseeded at equal densities in the top chamber of Matrigel™ invasion wellsand allowed to incubate for 48 (BT549 and BT474) and 72 (MCF7) hours,after which cells that had digested Matrigel™ and migrated through the 8um pores were counted on the underside of the insert. Representative 20×images are presented. MCF7 cells transduced with sh742 and sh528 show a65% and 71% decrease in invasion. BT549 cells transduced with sh742 andsh528 showed a 60% and 40% decrease in invasion. BT474 cells transducedwith sh742 and sh528 show an 82% decrease in invasion. Each knockdownwas compared to shScramble controls. The invasive phenotype ofshQSOX-transduced MCF7 (d), BT549 (e) and BT474 (1) cells was rescued byexogenous incubation with catalytically active rhQSOX1. rhQSOX1 (AA)mutant is a mutant without enzymatic activity, generously provided byDr. Debbie Fass. Graphs represent average±standard deviation (MCF7,BT549 and BT474 n=3), significance *, P<0.05, ** P<0.005.

FIG. 11. Reduced expression of QSOX1 in MCF7 and BT549 cells leads to adecrease in functional MMP-9 activity. Gelatin zymography of a) MCF7 andb) BT549 conditioned media shows a decrease in MMP-9 homodimers (130kDa) and MMP-9 (92 kDa). The percent decrease in MMP-9 expression inMCF7 was: sh742: 70% (p=0.0171); sh528: 77% (P=0.0182), and in BT549was: sh742: 34% (P=0.0531); sh528:88% (P=0.0564) compared to shScramblecontrol. c) Western blots of total cell lysate from shRNA treated MCF7and BT549 probing for MMP-2 and -9 show insignificant changes comparedto shScramble control. Full images can be seen in Additional file S3. d)QPCR of QSOX1 transcripts and MMP-2 and -9 transcripts. The graphrepresents relative gene expression calculated as ΔΔC_(q) using GAPDH asthe endogenous reference gene. MMP-2—MCF7 sh742 (P=0.5294), sh528(P=0.2112); BT549 sh742 (P=0.0054), sh528 (P=0.0019). MMP-9—MCF7 sh742(P=0.3981), sh528 (P=0.3385); BT549 sh742 (P=0.4192), sh528 (P=0.0701).Average±standard deviation; significance was determined using aStudent's two-tailed t-Test.

FIG. 12. GOBO analyses of QSOX1 transcript expression among subtypes ofbreast cancer from over 1,800 cases. a) Box plot analysis of QSOX1 mRNAexpression in all tumors ER+ (n=1,225) and ER− tumors (n=395)(P<0.00001); b) Box plot analyses of QSOX1 expression among HU subtypes,Basal (n=357), HER2 (n=152), Luminal A (n=482), Luminal B (n=289),Normal-like (n=257) and unclassified (n=344), (P<0.00001. c) KaplanMeier analysis over 10 years of relapse free survival (RFS) in patientswith Luminal B breast cancer expressing high (red line) and low (grayline) QSOX1 mRNA; High (n=56), low (n=74), (P=0.00062) and d) Overallsurvival (OS); High (n=34), low (n=64), (P=0.00031). Data obtained usingGOBO, Gene expression based Outcome for Breast cancer Online.

FIG. 13. Protein expression of QSOX1 is specific for breast tumor cellsin tissue. a) Graphical representation of “High QSOX1 staining (n=65)”column from Table 1. Each graph correlates with percentages of QSOX1positive cells listed in Table 1 for the “High QSOX1 staining (n=65)”column b) normal breast tissue showing no QSOX1 staining; c) grade 1invasive ductal carcinoma (IDC) ER+ PR+ breast tumor tissue showing lowQSOX1 staining; d) grade 2 IDC ER+ PR+ breast tumor tissue showingmoderate QSOX1 staining; e.) grade 3 IDC, ER+, PR+ showing high QSOX1staining; f) grade 3 invasive lobular carcinoma (ILC), ER+, PR− showinghigh QSOX1 staining Polyclonal antibody recognizes residues 1-329 ofboth QSOX1-S and -L.

FIG. 14. Reduced expression of QSOX1 leads to a significant decrease intumor cell growth. a) Western blot showing weak expression of QSOX1 intransformed, but non-tumor-forming MCF10A and human breast ductalcarcinoma cell lines Luminal A-like (MCF7 and MDA-MB-453), LuminalB-like (ZR75 and BT474) and Basal-like (BT549 and MDA-MB-231). α-Tubulinloading control is shown below each lane. MCF7, BT549 and BT474 breasttumor cell lines were transduced with lentiviral shRNA QSOX1 (sh742 andsh528). Western blots are shown using the same anti-QSOX1 polyclonal Abas in FIG. 2 on cell lysates from b) MCF7 (percent decrease in sh742:85% and sh528: 82%); c) BT549 (percent decrease in sh742: 45% and sh528:77%) and d) BT474 (percent decrease in sh742: 40% and sh528 36%) cells.Western blots have been cropped. (e-g) MTT and (h-j) Trypan Blue growthassays on MCF7, BT549 and BT474 cells transduced with shScramble, sh742and sh528 assayed on Days 1 through 5. Percent decrease sh742 and sh528day 5: e) 66% (both); f) 78% and 69%; g) 52% and 34%; h) 72% and 73%; i)98% and 96%; j) 50% (both). Experiments were performed three times intriplicate; error bars represent standard deviation from triplicatewells. Significance **, P<0.01.

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides methods for tumortreatment, comprising contacting a subject having a tumor with an amounteffective of an inhibitor of QSOX1 expression and/or activity, orpharmaceutically acceptable salt thereof, to treat the tumor.

As demonstrated in the examples that follow, the inventors of thepresent invention have discovered that inhibitors of QSOX1 expressionand/or activity of QSOX1 can be used to treat tumors.

As used herein, “QSOX1” is Quiescin Sulfhydryl Oxidase 1, also calledQSCN6. The protein accession number for the long variant of QSOX1 on theNCBI database is NP_002817 (SEQ ID NO:24), and the accession number forthe short form is NP_001004128 (SEQ ID NO:25). As used herein, “QSOX1”refers to both the long and short variants of QSOX1.

The subject can be any mammal, preferably a human.

As used herein, any suitable “inhibitor of QSOX1 expression and/oractivity” can be used that is capable of reducing expression of QSOX1mRNA expression or protein synthesis (including, but not limited toinhibiting QSOX1 promoter activity or reducing stability of QSOX1 mRNA),or that can inhibit activity of QSOX1 protein via any mechanism,including but not limited to binding to QSOX1 resulting in inhibition ofQSOX1 activity. Such inhibitors can include, but are not limited tosmall molecules, antibodies, and aptamers that inhibit activity of QSOX1protein, and antisense, siRNA, shRNA, etc. that inhibit expression ofQSOX1 mRNA and/or protein Inhibitors of QSOX1 expression may beidentified through any suitable means, including but not limited to themethods described below. Any suitable method for determining QSOX1activity levels may be used, including but not limited to thosedescribed in detail below.

As used herein, “inhibit” means at least a 10% reduction in QSOX1expression and/or activity; preferably at least a 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or greater reduction in expression and/or activity.

The methods of the invention can be used to treat any suitable tumortype. In one preferred embodiment, the methods are used to treat anytumor type that over-expresses QSOX1. Expression of QSOX1 can beassessed by any suitable method, including but not limited toimmunohistochemistry of suitable tissue sample, polymerase chainreaction, or detection of QSOX1 peptides in suitable tissue, asdisclosed, for example, in WO 2010/071788; WO 2010/01787; and WO2010/077921, incorporated by reference herein in their entirety. Invarious non-limiting embodiments, techniques that can be used in theanalysis include mass spectrometry (MS), two dimensional gelelectrophoresis, western blotting, immunofluorescence, ELISAs, antigencapture assays (including dipstick antigen capture assays) and mass specimmunoassay (MSIA). In one preferred embodiment, ligands for the one ormore peptides are used to “capture” antigens out of the tissue sample.Such ligands may include, but are not limited to, antibodies, antibodyfragments and aptamers. In one embodiment, the ligand(s) are immobilizedon a surface and the sample is passed over the surface under conditionssuitable for binding of any peptides in the sample to the ligand(s)immobilized on the surface. Such antigen capture assays permitdetermining a concentration of the peptides in the tissue sample, as theconcentration likely correlates with extent of disease.

The tissue sample may be any suitable sample from which tumor-derivedpeptides may be obtained. In various preferred embodiments, the tissuesample is selected from the group consisting of plasma, serum, urine,saliva, and relevant tumor tissue. In various preferred embodiments fordetecting QSOX1 peptides in tissue (preferably plasma or serum), thepeptide to be detected is selected from the group consisting of

(SEQ ID NO: 3) NEQEQPLGQWHLS, (SEQ ID NO: 4) NEQEQPLGQWH, (SEQ ID NO: 5)EQPLGQWHLS, (SEQ ID NO: 6) AAPGQEPPEHMAELQR, (SEQ ID NO: 7)AAPGQEPPEHMAELQ, (SEQ ID NO: 8) AAPGQEPPEHMAELQRNEQEQPLGQWHLS, (SEQ IDNO: 9) NEQEQPL, and (SEQ ID NO: 10) GQWHLS.

As used herein, the phrase “an amount effective” refers to the amount ofinhibitor that provides a suitable treatment effect.

Any suitable control can be used to compare with QSOX1 expression and/oractivity in the subject's tissue. In one embodiment, the controlcomprises an amount of one or more peptides of interest from a tissuesample from a normal subject or population (i.e. known not to besuffering from a tumor), or from a subject or population of subjectssuffering from a tumor, using the same detection assay. The controltissue sample will be of the same tissue sample type as that assessedfrom the test subject. In one preferred embodiment, a standardconcentration curve of one or more peptides of interest in the controltissue sample is determined, and the amount of the one or more peptidesof interest in the test subject's tissue sample is compared based on thestandard curve. In another preferred embodiment for use in monitoringprogress of tumor therapy, samples are obtained from patients over time,during or after their therapy, to monitor levels of one or more peptidesin plasma as an indication about tumor burden in patients. The controlmay comprise a time course of concentration of the one or more peptidesof interest in a given tissue type of the test subject, to monitor theeffect of treatment on the concentration; this embodiment is preferred,for example, when assessing efficacy of tumor treatments. Those of skillin the art will recognize that similar controls can be used forimmunohistochemical-based analysis. Based on the teachings herein andthe knowledge in the art, those of skill in the art can design a varietyof other appropriate controls in assessing QSOX1 expression and/oractivity in identifying subjects most likely to respond to the treatmentmethods of the invention, as well as to assess efficacy of the treatmentover time.

Thus, in one preferred embodiment, the method comprises identifyingsubjects with tumors that over-express QSOX1, and treating such patientsaccording to the methods of the invention. In one preferred embodiment,the method comprises measuring QSOX1 expression in blood plasma toidentify tumors that over-express QSOX1. Methods for preparing bloodplasma are well known in the art. In one embodiment, plasma is preparedby centrifuging a blood sample under conditions suitable for pelletingof the cellular component of the blood.

Non-limiting tumor types that can be treated using the methods of theinvention include pancreatic, lung, colon, breast, and prostate tumors.In one embodiment, the tumor is a pancreatic tumor, such as a pancreaticadenocarcinoma or a neuroendocrine tumor. In a further embodiment, thetumor comprises a pancreatic adenocarcinoma.

In a further embodiment, the tumor is a breast tumor. In one suchembodiment, the breast tumor is an estrogen receptor positive (ER+)breast tumor. In another embodiment, the breast tumor is a Luminal Btumor. For example, the Luminal B tumor is ER+ and/or progesteronereceptor positive (PR+). In another example, the Luminal B tumor ishighly positive for Ki67 and/or HER2-positive. In another example, theLuminal B tumor is ER+, PR−, and HER2-positive. In another example, theLuminal B tumor is ER+, PR+, and HER2-positive. In these embodiments,the methods may further comprise treating the patient with one or moreof chemotherapy, hormone therapy, and treatments targeted to HER2(including but not limited to pertuzumab, lapatinib, and trastuzumabemtansine (T-DM1)).

As used herein, “treating tumors” means accomplishing one or more of thefollowing: (a) reducing tumor mass; (b) slowing the increase in tumormass; (c) reducing size of tumor metastases and/or budding off ofmetastases; (d) slowing the incidence of tumor metastases; (e) limitingor preventing development of symptoms characteristic of cancer; (f)inhibiting worsening of symptoms characteristic of cancer; (g) limitingor preventing recurrence of symptoms of cancer in subjects that werepreviously symptomatic; (i) increasing subject survival time; and (j)limiting or reducing morbidity of therapy by enhancing current therapiesand/or permitting decreased dose of current standard of care therapies.

For example, symptoms of pancreatic cancer include, but are not limitedto, pain in the upper abdomen, significant weight loss, loss of appetiteand/or nausea and vomiting, jaundice, and Trousseau sign. Symptoms ofbreast cancer include, but are not limited to, a breast lump orthickening that feels different from the surrounding tissue, bloodydischarge from the nipple, change in the size or shape of a breast,changes to the skin over the breast, such as dimpling, inverted nipple,peeling, scaling or flaking of the nipple or breast skin; and redness orpitting of the skin over the breast.

In a preferred embodiment, the methods limit tumor metastasis, such aslimiting pancreatic or breast tumor metastasis. As shown in the examplesthat follow, the inventors have shown that knockdown of QSOX1 expressionin tumor cells leads to a dramatic decrease in tumor cellinvasive/migratory phenotype, thus making the methods of the inventionparticularly useful for limiting tumor metastasis. Silencing of QSOX1protein expression with shRNA was shown to inhibit breast tumor growthin vitro, and to suppress breast tumor cell invasion through Matrigel™(which can be rescued by addition of exogenous recombinant QSOX1).

While not being limited by any particular mechanism of action, theinventors believe that this inhibition of metastasis may result from adecrease in the proteolytic activity of matrix metalloproteases 2 and 9(MMP-2 and MMP-9), as discussed in more detail in the examples thatfollow.

In a preferred embodiment of all of the above embodiments, the inhibitoris selected from the group consisting of antibodies, antisense RNA,siRNA, miRNA, and shRNA. In a further preferred embodiment, theinhibitor comprises or consists of antisense, siRNA, miRNA, and/or shRNAmolecules having a nucleic acid sequence perfectly complementary toleast 10 contiguous nucleotides of QSOX1 as shown in SEQ ID NO:1 and SEQID NO:2 or RNA equivalents thereof; and/or fragments of the nucleic acidmolecule. In various preferred embodiments, the nucleic acid molecule isperfectly complementary to at least 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25 or more contiguous nucleotides of QSOX1, or anRNA equivalent thereof.

In another preferred embodiment, the inhibitor comprises or consists ofa nucleic acid selected from the group consisting of

(SEQ ID NO: 11) 5′-A(T/U)C(T/U)ACA(T/U)GGC(T/U)GACC(T/U)GGAA-3′ (SEQ IDNO: 12) 5′-AGGAAAGAGGG(T/U)GCCG(T/U)(T/U)C(T/U)(T/U)-3′, (SEQ ID NO: 13)5′-GCCAA(T/U)G(T/U)GG(T/U)GAGAAAG(T/U)(T/U)(T/U)- 3′, (SEQ ID NO: 14)5′-GCCAAGAAGG(T/U)GAAC(T/U)GGA(T/U)(T/U)-3′, (SEQ ID NO: 15)5′-CCGGACAA(T/U)GAAGAAGCC(T/U)(T/U)(T/U)-3′, and (SEQ ID NO: 16)5′-(T/U)C(T/U)AGCCACAACAGGG(T/U)CAA(T/U)-3′.

wherein residues noted as “(T/U)” can be either “T” or “U’. In furtherpreferred embodiments, the inhibitor comprises or consists of

(SEQ ID NO: 17) 5′-ATCTACATGGCTGACCTGGAA-3′, (SEQ ID NO: 18)5′-AGGAAAGAGGGTGCCGTTCTT-3′, (SEQ ID NO: 19)5′-GCCAATGTGGTGAGAAAGTTT-3′, (SEQ ID NO: 20)5′-GCCAAGAAGGTGAACTGGATT-3′, (SEQ ID NO: 21)5′-CCGGACAATGAAGAAGCCTTT-3′; (SEQ ID NO: 22)5′-TCTAGCCACAACAGGGTCAAT-3′; and (SEQ ID NO: 26)5′-CCGGGCCAATGTGGTGAGAAAGTTTCTCGAGAAACTTT CTCACCACATTGGCTTTTTG-3′.

In a further preferred embodiment, the nucleic acids of SEQ ID NO:11-22are part of a short hairpin RNA (shRNA). In this embodiment, such anshRNA comprises flanking regions, loops, and antisense and spacersequences flanking a recited SEQ ID NO: sequence responsible for thespecificity of shRNA. There are no specific sequence requirements forthese various other shRNA regions so long as a loop structure can beformed. Exemplary constructs are shown in the examples below. ShRNA arethought to assume a stem-loop structure with a 2 nucleotide 3′ overhangthat is recognized by Dicer and processed in to small interfering RNA(siRNA). siRNA are then recognized by RNA-Induced Silencing Complex(RISC), which removes the sense strand from the stem structure andleaves the guide strand to associate with target mRNA, QSOX1 mRNA inthis case, and cleave it. Full length protein cannot be translated aftercleavage.

In one exemplary embodiment, the shRNA comprise or consist of a nucleicacid of the general formula:

(SEQ ID NO: 23) CCGG-X1-CTCGAGAAACTTTCTCACCACATTGGCTTTTTG-3′

wherein X1 is a nucleic acid sequence selected from the group consistingof

(SEQ ID NO: 11) 5′-A(T/U)C(T/U)ACA(T/U)GGC(T/U)GACC(T/U)GGAA-3′ (SEQ IDNO: 12) 5′-AGGAAAGAGGG(T/U)GCCG(T/U)(T/U)C(T/U)(T/U)-3′, (SEQ ID NO: 13)5′-GCCAA(T/U)G(T/U)GG(T/U)GAGAAAG(T/U)(T/U)(T/U)- 3′, (SEQ ID NO: 14)5′-GCCAAGAAGG(T/U)GAAC(T/U)GGA(T/U)(T/U)-3′. (SEQ ID NO: 15)5′-CCGGACAA(T/U)GAAGAAGCC(T/U)(T/U)(T/U)-3′ (SEQ ID NO: 16)5′-(T/U)C(T/U)AGCCACAACAGGG(T/U)CAA(T/U)-3′ (SEQ ID NO: 17)5′-ATCTACATGGCTGACCTGGAA-3′, (SEQ ID NO: 18)5′-AGGAAAGAGGGTGCCGTTCTT-3′, (SEQ ID NO: 19)5′-GCCAATGTGGTGAGAAAGTTT-3′, (SEQ ID NO: 20)5′-GCCAAGAAGGTGAACTGGATT-3′, (SEQ ID NO: 21)5′-CCGGACAATGAAGAAGCCTTT-3′; and (SEQ ID NO: 22)5′-TCTAGCCACAACAGGGTCAAT-3′.

These and other nucleic acid inhibitors may be modified for a desiredpurpose, including but not limited to nucleic acid backbone analoguesincluding, but not limited to, phosphodiester, phosphorothioate,phosphorodithioate, methylphosphonate, phosphoramidate, alkylphosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino),3′-N-carbamate, morpholino carbamate, peptide nucleic acids (PNAs),methylphosphonate linkages or alternating methylphosphonate andphosphodiester linkages (Strauss-Soukup (1997) Biochemistry36:8692-8698), and benzylphosphonate linkages, as discussed in U.S. Pat.No. 6,664,057; see also Oligonucleotides and Analogues, a PracticalApproach, edited by F. Eckstein, IRL Press at Oxford University Press(1991); Antisense Strategies, Annals of the New York Academy ofSciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan(1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications(1993, CRC Press). Nucleic acid inhibitors may also comprise analogousforms of ribose or deoxyribose as are well known in the art, includingbut not limited to 2′ substituted sugars such as 2′-O-methyl-,2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, α.-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, sedoheptuloses, acyclic analogs and a basic nucleoside analogssuch as methyl riboside. The oligonucleotides may also contain TNA(threose nucleic acid; also referred to as alpha-threofuranosyloligonucleotides) (See, for example, Schong et al., Science 2000 Nov.17, 290 (5495):1347-1351.)

The inhibitors for use in the present invention can be administered viaany suitable technique or formulation, including but not limited tolipid, virus, polymer, or any other physical, chemical or biologicalagent, but are generally administered as part of a pharmaceuticalcomposition together with a pharmaceutically acceptable carrier,diluent, or excipient. Such compositions are substantially free ofnon-pharmaceutically acceptable components, i.e., contain amounts ofnon-pharmaceutically acceptable components lower than permitted by USregulatory requirements at the time of filing this application. In someembodiments of this aspect, if the compound is dissolved or suspended inwater, the composition further optionally comprises an additionalpharmaceutically acceptable carrier, diluent, or excipient. In otherembodiments, the pharmaceutical compositions described herein are solidpharmaceutical compositions (e.g., tablet, capsules, etc.). The isolatednucleic acids or shRNAs can be present in a vector, such as a viralvector (ex: retrovirus, lentivirus, adenovirus, adeno-associated virus,etc.), for delivery via any suitable technique.

These compositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be via physical injectionwith a needle to, for example, a tumor in the subject; topical(including ophthalmic and to mucous membranes including intranasal,vaginal and rectal delivery), pulmonary (e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal), ocular, oral orparenteral. Methods for ocular delivery can include topicaladministration (eye drops), subconjunctival, periocular or intravitrealinjection or introduction by balloon catheter or ophthalmic insertssurgically placed in the conjunctival sac. Parenteral administrationincludes intravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration. Parenteral administration can be inthe form of a single bolus dose, or may be, for example, by a continuousperfusion pump. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Also, pharmaceutical compositions can contain, as the active ingredient,one or more inhibitors described herein above in combination with one ormore pharmaceutically acceptable carriers, and may further comprise oneor more additional active agents as appropriate for a given therapeutictreatment. In making the compositions described herein, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions described herein can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 1 to about 1000 mg, more usually about 10 to about500 mg, of the active ingredient. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient. It will beunderstood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual inhibitor administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

The inhibitors described herein can also be formulated in combinationwith one or more additional active ingredients as desired.

The present invention also provides methods for limiting tumormetastasis, comprising contacting a subject having a tumor with anamount effective of an inhibitor of QSOX1 expression and/or activity, orpharmaceutically acceptable salt thereof, to limit metastasis of thetumor in the subject. As shown in the examples that follow and asdiscussed above, QSOX1 inhibitors slow tumor growth and inhibit themetastatic process.

All embodiments of the first aspect of the invention are equallyapplicable to this second aspect, unless the context clearly dictatesotherwise. As used herein, “limiting metastasis” means any limitationover what would be seen in the absence of administration of the QSOX1inhibitor. In a preferred embodiment, limiting metastasis comprises astatistically significant limitation compared to control subjects nottreated with the QSOX1 inhibitor. In another preferred embodiment, thetumor comprises a pancreatic tumor; even more preferably a pancreaticadenocarcinoma.

In a second aspect, the present invention provides isolated nucleicacids, comprising or consisting of antisense, siRNA, miRNA, and/or shRNAmolecules having a nucleic acid sequence perfectly complementary toleast 10 contiguous nucleotides of QSOX1 as shown in SEQ ID NO:1 and SEQID NO:2 or RNA equivalents thereof; and/or fragments of the nucleic acidmolecule. In various preferred embodiments, the nucleic acid molecule isperfectly complementary to at least 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25 or more contiguous nucleotides of QSOX1, or anRNA equivalent thereof.

In a further preferred embodiment, the isolated nucleic acids compriseor consist of a nucleotide sequence selected from the group consistingof

(SEQ ID NO: 11) 5′-A(T/U)C(T/U)ACA(T/U)GGC(T/U)GACC(T/U)GGAA-3′ (SEQ IDNO: 12) 5′-AGGAAAGAGGG(T/U)GCCG(T/U)(T/U)C(T/U)(T/U)-3′, (SEQ ID NO: 13)5′-GCCAA(T/U)G(T/U)GG(T/U)GAGAAAG(T/U)(T/U)(T/U)- 3′, (SEQ ID NO: 14)5′-GCCAAGAAGG(T/U)GAAC(T/U)GGA(T/U)(T/U)-3′. (SEQ ID NO: 15)5′-CCGGACAA(T/U)GAAGAAGCC(T/U)(T/U)(T/U)-3′ (SEQ ID NO: 16)5′-(T/U)C(T/U)AGCCACAACAGGG(T/U)CAA(T/U)-3′ (SEQ ID NO: 17)5′-ATCTACATGGCTGACCTGGAA-3′, (SEQ ID NO: 18)5′-AGGAAAGAGGGTGCCGTTCTT-3′, (SEQ ID NO: 19)5′-GCCAATGTGGTGAGAAAGTTT-3′, (SEQ ID NO: 20)5′-GCCAAGAAGGTGAACTGGATT-3′, (SEQ ID NO: 21)5′-CCGGACAATGAAGAAGCCTTT-3′; (SEQ ID NO: 22)5′-TCTAGCCACAACAGGGTCAAT-3′; and (SEQ ID NO: 26)5′-CCGGGCCAATGTGGTGAGAAAGTTTCTCGAGAAACTTT CTCACCACATTGGCTTTTTG-3′.

The isolated nucleic acids can be used, for example, in the methods ofthe invention. The isolated nucleic acids of this second aspect of thepresent invention can be modified as described above in the first aspectof the invention, including nucleic acid backbone analogues andanalogous forms of ribose or deoxyribose.

As used herein, “isolated” means that the nucleic acids are removed fromtheir normal surrounding nucleic acid sequences in the genome or in cDNAsequences, and are substantially free of contaminating material used toisolate them (i.e. agarose, polyacrylamide, column chromatographyresins, and the like). The isolated nucleic acids may be stored in anysuitable state, including but not limited to in solution or as alyophilized powder.

The isolated nucleic acids may be chemically synthesized using meansknown in the art, or may be generated by recombinant expression vectors.

As used herein, the isolated nucleic acids “comprising” the recitednucleotide sequences means that the recited nucleotide sequences can bepresent as part of a larger synthetic construct, such as an antisensenucleic acid, siRNA, an shRNA, miRNA, or as part of a construct inassociation (covalently bound or non-covalently bound) with a lipid,virus, polymer, or any other physical, chemical or biological agent. Asused herein, the isolated nucleic acids “comprising” the recitednucleotide sequences does not include the isolated nucleic acid as partof a naturally occurring or isolated full length QSOX1 transcript orcDNA thereof.

In one embodiment, the isolated nucleic acid is present in a shorthairpin RNA (shRNA). As discussed above, such an shRNA comprisesflanking regions, loops, antisense and spacer sequences flanking arecited SEQ ID NO: sequence responsible for the specificity of shRNA.There are no specific sequence requirements for these various othershRNA regions so long as a loop structure can be formed. Exemplaryconstructs are shown in the examples below. In one embodiment, the shRNAis of the general formula:

(SEQ ID NO: 23) CCGG-X1-CTCGAGAAACTTTCTCACCACATTGGCTTTTTG-3′

wherein X1 is a nucleic acid sequence selected from the group consistingof

(SEQ ID NO: 11) 5′-A(T/U)C(T/U)ACA(T/U)GGC(T/U)GACC(T/U)GGAA-3′ (SEQ IDNO: 12) 5′-AGGAAAGAGGG(T/U)GCCG(T/U)(T/U)C(T/U)(T/U)-3′, (SEQ ID NO: 13)5′-GCCAA(T/U)G(T/U)GG(T/U)GAGAAAG(T/U)(T/U)(T/U)- 3′, (SEQ ID NO: 14)5′-GCCAAGAAGG(T/U)GAAC(T/U)GGA(T/U)(T/U)-3′. (SEQ ID NO: 15)5′-CCGGACAA(T/U)GAAGAAGCC(T/U)(T/U)(T/U)-3′ (SEQ ID NO: 16)5′-(T/U)C(T/U)AGCCACAACAGGG(T/U)CAA(T/U)-3′ (SEQ ID NO: 17)5′-ATCTACATGGCTGACCTGGAA-3′, (SEQ ID NO: 18)5′-AGGAAAGAGGGTGCCGTTCTT-3′, (SEQ ID NO: 19)5′-GCCAATGTGGTGAGAAAGTTT-3′, (SEQ ID NO: 20)5′-GCCAAGAAGGTGAACTGGATT-3′, (SEQ ID NO: 21 5′-CCGGACAATGAAGAAGCCTTT-3′;and (SEQ ID NO: 22) 5′-TCTAGCCACAACAGGGTCAAT-3′.

In a third aspect, the present invention provides recombinant expressionvectors comprising the isolated nucleic acid of any embodiment of thethird aspect of the invention operatively linked to a promoter.“Recombinant expression vector” includes vectors that operatively link anucleic acid coding region or gene to any promoter capable of effectingexpression of the gene product. The vectors can be used, for example,for transfection of host cells for large scale production of theisolated nucleic acids, or may be used as vector delivery systems in themethods of the invention. The promoter sequence used to drive expressionof the disclosed nucleic acid sequences in a mammalian system may beconstitutive (driven by any of a variety of promoters, including but notlimited to, utilizes the U6 or H1 promoter promoter (to ensure that theshRNA is always expressed), CMV, SV40, RSV, actin, EF) or inducible(driven by any of a number of inducible promoters including, but notlimited to, tetracycline, ecdysone, steroid-responsive). Theconstruction of expression vectors for use in transfecting eukaryoticand prokaryotic cells is also well known in the art, and thus can beaccomplished via standard techniques. (See, for example, Sambrook,Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1989; Gene Transfer and ExpressionProtocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc.,Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.). Theexpression vector must be replicable in the host organisms either as anepisome or by integration into host chromosomal DNA. In a preferredembodiment, the expression vector comprises a viral vector or a plasmid.Any suitable viral vector may be used, including but not limited toretroviruses, lentiviruses, adenoviruses, adeno-associated viruses, etc.

In a fourth aspect, the present invention provides host cells comprisingthe recombinant expression vectors disclosed herein, wherein the hostcells can be either prokaryotic or eukaryotic. The host cells can beused, for example, in large scale production of the recombinant vectorsof the invention. The cells can be transiently or stably transfected ifa plasmid vector is used, or may be transiently or stably transducedwhen a viral vector is used. Such transfection and transduction ofexpression vectors into prokaryotic and eukaryotic cells can beaccomplished via any technique known in the art, including but notlimited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome-mediated, DEAEdextran-mediated, polycationic mediated, or viral mediated transfection.(See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, etal., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells:A Manual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss,Inc. New York, N.Y.).

In a fifth aspect, the present invention provides pharmaceuticalcompositions, comprising

(a) the isolated nucleic acid of any embodiment of the second aspect ofthe invention, the recombinant expression vector of any embodiment ofthe third aspect of the invention, or the recombinant host cell of anyembodiment of the fourth aspect of the invention; and

(b) a pharmaceutically acceptable carrier.

The pharmaceutical compositions can be used, for example, in the methodsof the invention. As used herein, the phrase “pharmaceuticallyacceptable salt” refers to both pharmaceutically acceptable acid andbase addition salts and solvates. Such pharmaceutically acceptable saltsinclude salts of acids such as hydrochloric, phosphoric, hydrobromic,sulfuric, sulfinic, formic, fumaric, toluenesulfonic, methanesulfonic,nitric, benzoic, citric, tartaric, maleic, hydroiodic, alkanoic such asacetic, HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like. Non-toxicpharmaceutical base addition salts include salts of bases such assodium, potassium, calcium, ammonium, and the like. Those skilled in theart will recognize a wide variety of non-toxic pharmaceuticallyacceptable addition salts. All embodiments of pharmaceuticalcompositions discussed herein for the methods of the invention can beused in this aspect of the invention.

In a sixth aspect, the present invention provides methods foridentifying candidate compounds for treating a tumor, comprising

(a) contacting tumor cells capable of expressing QSOX1 with one or morecandidate compounds under conditions suitable for expression of QSOX1;

(b) determining a level of QSOX1 expression and/or activity in the tumorcells and comparing to control;

wherein a compound that decreases QSOX1 expression and/or activity inthe tumor cells relative to control is a candidate compound for treatinga tumor.

Any tumor cell that is capable of expressing QSOX1, either inherently oras a result of transfecting the cell with a QSOX1 recombinant expressionvector, can be used. In a preferred embodiment, the tumor cell isselected from the group consisting of pancreatic, lung, colon, breast,and prostate tumor cells. In one embodiment, the tumor cells arepancreatic tumor cells, such as pancreatic adenocarcinoma cells. Inanother embodiment, the tumor cells are breast tumor cells, such as ER+breast cancer cells or luminal B cancer cells.

Any suitable candidate compound can be used, including but not limitedto small molecules, antibodies, aptamers, antisense, siRNA, and shRNA.

As used herein, “inhibit” means at least a 10% reduction in expressionand/or activity; preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or greater reduction in expression and/or activity.

Any suitable control can be used, including but not limited to tumorcells not treated with test compound, and average expression productlevels of QSOX1 in a control cell population. In one embodiment, thecontrol comprises a cell from a normal subject or population (i.e. knownnot to be suffering from a tumor), or from a subject or population ofsubjects suffering from a tumor, using the same detection assay. Inanother preferred embodiment, the control comprises a tumor cellcontacted with the isolated nucleic acid, shRNA, or expression vector ofthe invention, to facilitate identifying compounds with increased QSOX1inhibitory activity than the nucleic acids disclosed herein. The controlcell will be of the same type as that assessed from the test subject. Inone exemplary embodiment where immunohistochemistry is used to assessQSOX1 expression, a suitable control is normal tissue in a pathologicalsample.

Any suitable method for determining QSOX1 expression levels may be used,including but not limited to reverse transcription-polymerase chainreaction (RT-PCR), western blot, in situ hybridization, andimmunohistochemical analysis (such as fluorescence in situhybridization)

Similarly, any suitable method for determining QSOX1 activity levels maybe used, including but not limited to an oxygen electrode assay, usingthe enzymatic activity of QSOX1 to detect structures/compounds thatinhibit the ability of QSOX1 to oxidize a known substrate.

In another aspect, the invention provides methods for prognosing atumor, comprising

(a) determining a QSOX1 expression level in a sample from a subjecthaving a tumor;

(b) comparing the QSOX1 expression level to control; and

(c) prognosing the progression of the tumor in the subject.

As shown in the examples that follow, subjects with tumors thatover-express QSOX1 have a poorer prognosis than patients that do notover-express QSOX1. As shown in the examples that follow, QSOX1 isassociated with a highly invasive tumor phenotype and correlates withpoor prognosis, such as risk of relapse and poor survival in patientswith tumors, such as breast tumors, and particularly Luminal B breasttumors. As further shown in the examples, QSOX1 overexpressioncorrelates with increasing tumor grade, and is independent of and notassociated with Her2 expression or cytokeratin 5/6 expression.

As used herein, “over-express” means at least a 10% increase in QSOX1mRNA or protein expression compared to control; in various otherembodiments, at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, orgreater increase in expression.

Thus, the methods can be used to determine a likely prognosis for agiven subject, and thus allows the subject and attending physician totailor treatment accordingly. Thus, in a further embodiment, the methodscomprise determining a course of treatment based in part on theprognosis.

The tissue sample may be any suitable sample from which tumor-derivedQSOX1 peptides or mRNA may be obtained. In various embodiments, thetissue sample is selected from the group consisting of plasma, serum,urine, saliva, and relevant tumor tissue. In various embodiments fordetecting QSOX1 peptides in tissue (preferably plasma or serum), thepeptide to be detected is, or the mRNA to be detected encodes, one ormore peptide selected from the group consisting of

(SEQ ID NO: 3) NEQEQPLGQWHLS, (SEQ ID NO: 4) NEQEQPLGQWH, (SEQ ID NO: 5)EQPLGQWHLS, (SEQ ID NO: 6) AAPGQEPPEHMAELQR, (SEQ ID NO: 7)AAPGQEPPEHMAELQ, (SEQ ID NO: 8) AAPGQEPPEHMAELQRNEQEQPLGQWHLS, (SEQ IDNO: 9) NEQEQPL, and (SEQ ID NO: 10) GQWHLS.

Any suitable control can be used to compare with QSOX1 expression and/oractivity in the subject's sample. In one embodiment, the controlcomprises an amount of one or more peptides or mRNAs of interest from atissue sample from a normal subject or population (i.e.: known not to besuffering from a tumor), or from a subject or population of subjectssuffering from a tumor, using the same detection assay. The controltissue sample will be of the same tissue sample type as that assessedfrom the test subject. In one embodiment, a standard concentration curveof one or more peptides or mRNAs of interest in the control tissuesample is determined, and the amount of the one or more peptides ormRNAs of interest in the test subject's tissue sample is compared basedon the standard curve. In another embodiment samples are obtained frompatients over time, during or after their therapy, to monitor levels ofone or more peptides or mRNAs in plasma as an indication about tumorburden in patients. The control may comprise a time course ofconcentration of the one or more peptides or mRNAs of interest in agiven tissue type of the test subject, to monitor the effect oftreatment on the concentration; this embodiment is preferred, forexample, when assessing efficacy of tumor treatments. Those of skill inthe art will recognize that similar controls can be used forimmunohistochemical-based analysis. Based on the teachings herein andthe knowledge in the art, those of skill in the art can design a varietyof other appropriate controls in assessing QSOX1 expression.

Non-limiting tumor types that can be prognosed using the methods of theinvention include pancreatic, lung, colon, breast, and prostate tumors.In one embodiment, the tumor is a pancreatic tumor, such as a pancreaticadenocarcinoma or a neuroendocrine tumor. In a further embodiment, thetumor comprises a pancreatic adenocarcinoma. In a further embodiment,the tumor is a breast tumor. In one such embodiment, the breast tumor isan estrogen receptor positive (ER+) breast tumor. In another embodiment,the breast tumor is a Luminal B tumor. For example, the Luminal B tumoris ER+ and/or progesterone receptor positive (PR+). In another example,the Luminal B tumor is highly positive for Ki67 and/or HER2-positive. Inanother example, the Luminal B tumor is ER+, PR−, and HER2-positive. Inanother example, the Luminal B tumor is ER+, PR+, and HER2-positive.

As used herein, “prognosing the progression of a tumor” means assessingone or more of the following: (a) aggressiveness of tumor growth; (b)likelihood of tumor metastases; (c) likelihood of therapeutic benefit ofa treatment course; (d) risk of tumor relapse/recurrence post-treatment;and (e) patient survival.

Example 1

Pancreatic ductal adenocarcinoma (PDA) is a disease that carries a poorprognosis. It is often detected in stage III resulting in anunresectable tumor at the time of diagnosis. However, even if pancreaticcancer is surgically resected in stage I or II, it may recur at ametastatic site (1, 2). Currently, patients diagnosed with pancreaticductal adenocarcinoma have less than a 5% chance of surviving past fiveyears (3). Through proteomic analysis of pancreatic cancer patientplasma, we discovered a peptide from QSOX1 that maps back to theC-terminus of the long isoform of QSOX1 (QSOX1-L) (4). Subsequently, wefound that QSOX1 is over-expressed in tumor tissue from pancreaticcancer patients, but not adjacent normal tissue (FIGS. 1B & C). Thesefindings led us to hypothesize that over-expression of QSOX1 might befunctionally important for tumor cells, prompting further exploration ofthe role that QSOX1 might play in pancreatic cancer.

QSOX1 belongs to the family of FAD-dependent sulfhydryl oxidases thatare expressed in all eukaryotes sequenced to date. As eloquently shownby the Thorpe and Fass laboratories, the primary enzymatic function ofQSOX1 is oxidation of sulfhydryl groups during protein folding togenerate disulfide bonds in proteins, ultimately reducing oxygen tohydrogen peroxide (5-7). QSOX1 has been reported to be localized to theGolgi apparatus and endoplasmic reticulum (ER) in human embryonicfibroblasts where it works with protein disulfide isomerase (PDI) tohelp fold nascent proteins in the cell (8, 9).

In the human genome, QSOX1 is located on chromosome 1q24 and alternativesplicing in exon 12 generates a long (QSOX1-L) and short (QSOX1-S)transcript (FIG. 1A) (10). Both, QSOX1-S and -L have identicalfunctional domain organization from the amino terminus as follows: twothioredoxin-like domains (Trx1 &2), a helix rich region (HRR) and anErv/ALR FAD-binding domain (5, 11). QSOX1-L contains a predictedtransmembrane domain that is not present in QSOX1-S due to alternativesplicing (FIG. 1A) (12). QSOX1 was originally discovered in quiescenthuman lung fibroblasts and was hypothesized to aid in the transitionfrom G₀ to S phase of the cell cycle (13, 14). Thorpe et al. revealedthe ability of QSOX1 to efficiently generate disulfide bonds intoproteins during folding at rate of 1000 per minute with a K_(M) of 150uM per thiol (7). QSOX1 appears to play a significant role in redoxregulation within the cell, although the in vivo biological substratesare undefined as well as the functional significance associated witheach splice variant.

In the present study, we have begun to analyze the role of QSOX1 inpancreatic tumors using cell lines BxPC3 and Panc-1. We knocked downQSOX1-S and -L protein expression using short hairpin RNAs (shRNA) in anattempt to reveal how pancreatic cancer cells might be affected. Weassessed cell growth, cell cycle, apoptosis, invasion and matrixmetalloproteinase activity. QSOX1 knock-downs affected tumor cellproliferation, cell cycle and apoptosis. We observed a dramatic decreasein tumor cell invasion when QSOX1 expression was suppressed. Furtherinvestigation into the mechanism of invasion revealed that QSOX1 is atleast partially responsible for MMP-2 and MMP-9 activity. This is thefirst report demonstrating a role for QSOX1 in invasion and metastasis.

Material and Methods for Example 1 Cell Culture

Pancreatic adenocarcinoma BxPC3, PANC-1, CFPac-1, and Capan1 cancer celllines were cultured in DMEM with 10% fetal bovine serum (FBS) (Gibco).Immortal human non-tumorigenic pancreatic duct epithelial cells (HPDE6)were cultured in Clontech KGM-2 karotinocyte media (Gibco) (19). Allcell lines were grown at 37° C. with 5% CO₂. Cell lines are testedmonthly for mycoplasma contamination using, Venor GeM™ MycoplasmaDetection Kit, PCR based from Sigma.

Immunohistochemistry (IHC)

Immunohostochemistry on patients who underwent surgical resection wasperformed in the exact same manner as previously described in Kwasi etal (4).

Generation of Short Hairpin (Sh) RNA and Lentiviruses Production

Three different shRNA for QSOX1 were obtained through DNASU(http://dnasu.asu.edu)(20) already in the lentiviral pLKO.1-puromycinselection vector.

QSOX1 sh742, (SEQ ID NO: 26) 5′-CCGG GCCAATGTGGTGAGAAAGTTT CTCGAGAAACTTTCTCACCACATTGGCTTTTTG-3′ (sense), QSOX1 sh528, (SEQ ID NO: 21) 5′-CCGGACAATGAAGAAGCCTTT -3′ (sense), QSOX1 sh616, (SEQ ID NO: 22) 5′-TCTAGCCACAACAGGGTCAAT -3′ (sense) and shScramble with target sequence(SEQ ID NO: 29) 5′- TCCGTGGTGGACAGCCACATG -3′was obtained from Josh LaBaer's laboratory at Arizona State University.The target sequence is underlined and each vector contains the samesupporting sequence surrounding the target sequence.

Lentiviruses containing sh742, sh528, sh616 and shScramble were producedusing 293T cells. 293T cells were seeded at 1.5×10⁶ cells per well in 2mL media lacking antibiotics using a 6 well plate format and incubatedat 37° C., 5% CO₂ for 24 hrs. The following day the 293T cells weretransfected with 2500 ng shRNA maxi-prepped plasmid DNA (SigmaGeneElute™ HP Plasmid Maxiprep Kit), 500 ng VSVg, 2500 ng d8.91(gag-pol)in LT1 transfection reagent from Mirus Bio (Madison, Wis.) andcentrifuged at 1000 g for 30 minutes and incubated as 37° C., 5% CO₂ for24 hours at in media lacking antibiotics. The next morning mediacontaining lentivirus was collected and replaced with complete media.Supernatants (2.5 ml) from transfected 293T cells producing eachlentivirus were collected every 24 hours for a total of 72 hours,combined and stored at −20° C.

Generation of shQSOX1-Transduced Tumor Cell Lines

Stable transduction of sh742, sh528, sh616 and shScramble into BxPC-3and Panc-1 cell lines was performed by first seeding the cells at 8×10⁵cells/well in a 6 well plate and incubating overnight. The next day thecells were transduced by adding 8 ug/mL polybrene (Millipore) and 200 ulsh742, sh528, sh616 and shScramble lentivirus media from 293T cells toeach well. The cells were spun at 1000 rpm for 30 minutes and thenincubated for 24 hours. The following day fresh DMEM with 10% FBS wasadded, containing 1 ug/mL puromycin (Sigma), to select for thetransduced cells QSOX1 knockdown was measured by western blot.

SDS-PAGE-Western Blotting

Western blotting was performed using cell lysates from HPDE6, BxPC3,Panc-1, Capan1 and CFPac1 cells as well as patient 1010 and 1016 tumorand adjacent normal enzymatic supernatant. Cell lysates were generatedby harvesting 2.5×10⁶ cells by centrifugation followed by lysis usingRIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1%Triton X-100) with 1× SigmaFAST™ Protease Inhibitor Cocktail Tablet,EDTA Free. Protein in the cell lysate was measured using the micro BCAprotein assay kit (Thermo Scientific). All samples were then normalizedto 2 ug/mL (20 ug total protein per lane). Samples were run on 10%SDS-polyacrylamide gels then transferred onto Immun-Blot™ PVDF Membranes(Bio-Rad). Rabbit polyclonal anti-QSOX1 (ProteinTech), rabbit polyclonalanti-Bactin and anti-alpha-tubulin (Cell Signaling), and rabbitpolyclonal anti-MMP-2 and -9 (Sigma) antibody was diluted 1:1000,1:1000, and 1:500 respectfully, in 0.1% BSA in 1×TBS+0.01% Tween-20 andincubated for overnight. Goat anti-rabbit IgG-alkaline phospatase or HRPsecondary antibody was used at a 1:5000 dilution and incubated with theblot for 1 hour followed by washing. BCIP/NBT substrate (PierceChemical, Rockford, Ill.) was added and the blot was developed at roomtemperature (RT) for approximately 1 hour, in samples incubated inalkaline phosphatase secondary antibody. For samples incubated in goatanti-rabbit HRP secondary the blots were developed using Novex ECLChemiluminescent Substrate Reagent Kit. Quantification of band intensitywas measured using Image J and is presented as percent change from thescrambled shRNA control. Full gel images are available in thesupplemental material. All gel images were annotated and processed usingPhotoshop software.

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay

Cells were seeded at 3×10³ cell/well in a 96-well plate in triplicateand incubated at 37° C., 5% CO₂ over the course of 5 days. The MTT assaywas performed on days 1, 2 and 5 according to the manufacturers'instructions (Invitrogen-Molecular Probes, Vybrant MTT CellProliferation Assay Kit). Results are presented as average±standarddeviation. Student's two-tailed t-Test was performed to determinesignificance.

Annexin V/Propidium Iodide Apoptosis Analysis

Apoptosis analysis was performed according to the manufacturer'sinstructions (FITC Annexin V Apoptosis Detection Kit I, BD Pharmingen).Briefly, cells were seeded at equal densities in a 25 cm² flask untilthey reached 60-80% confluency. The cells were then washed with coldPBS, counted, and normalized to 1×10⁶ cell/ml in 1× Annexin V BindingBuffer. Next, 1×10⁵ cells were then transferred to a separate tube and 5ul of FITC Annexin V and 5 ul of Propidium Iodide were added to eachsample. The samples were gently vortexed and incubated for 15 min at RTin the dark. Lastly, 400 ul of 1× Binding Buffer was added to eachsample and the samples were analyzed by flow cytometer (Becton DickinsonFACScalibur™ Flowcytometer) with 1 hour. Each sample was performed intriplicate.

Gelatin Zymography

The identification of matrix metalloproteinases (MMP) was performedusing gelatin zymography. Zymography experiments were performed asfollows. Untreated BxPC3 and Panc-1 cells as well as transduced cellswere seeded at 5×10⁵ cells/well (12 well plates) in DMEM with 10% FBS.The next day, cells were then washed with 1×PBS and the media waschanged to serum free DMEM and incubated for 24 hours before beingcollected and protein concentrations determined using a BCA assay.Gelatin zymography was performed with a 10% polyacrylamide gelcontaining gelatin solution in place of water (0.8 mg/mL Gelatin, 0.15 MTris pH 8.8, 30% acrylamide-bis, 50% glycerol, 10% SDS, 10% APS, andTEMED) (21). A volume of equal concentrations of serum free conditionedmedia were loaded under non-denaturing conditions into the 10%polyacrylamide-gelatin gel to separate proteins secreted by the tumorcells and to detect the presence of gelatin degrading MMPs.Electrophoresis was performed at a constant voltage of 150 V for 60 min.Gels were washed in renaturing buffer (25% Triton X-100 in water) for 30min at RT with gentle shaking. The gels were then equilibrated indeveloping buffer (50 mM Tris-base, 6.3 g/L Tris-HCl, 0.2 M NaCl, 5 mMCaCl₂, and 0.02% Triton X-100) for 30 min at RT with gentle shakingFresh developing buffer was then added to the gels and they wereincubated overnight at 37° C. The gels were then stained withSimplyBlue™ Safe Stain (Invitrogen) for 20 min at RT, then destainedovernight in ddH₂O at RT. The presence of MMP was detected by the lackof staining indicating digestion of gelatin. The negative control wasperformed by adding, 50 mM Ethylene Diamine Tetra Acetic Acid (EDTA), toboth the renaturing buffer and the developing buffer to block the MMPactivation. Quantification of band intensity was measured using Image Jand is presented as percent change from the scrambled shRNA control.

RNA Isolation and cDNA Synthesis

Total RNA isolation was performed according to the manufacturesinstructions for animal cells using spin technology (RNeasy™ Mini Kit,Qiagen). After RNA was isolated from each sample was reverse transcribedwith gScript™ cDNA Sythesis Kit, Quanta Biosciences according to themanufactures instructions.

Quantitative Real Time PCR (qPCR)

The relative level of GAPDH, QSOX1-L, QSOX1-S, MMP-2, and MMP-9 wereanalyzed in each sample by qPCR. Each cDNA sample was normalized to 100ng/μl in molecular grade water along with 100 nM final concentration ofeach primer and 1× final concentration of PerfeCta™ SYBR Green Fast Mix,ROX to a final volume of 20 μl. qPCR was performed using, PerfeCTa™ SYBRGreen FastMix, ROX from Quanta Biosciences on a ABI7900HT thermocycler,Applied Biosystems Inc. Reaction Protocol: Initial Denaturation—95° C.for 3 min; PCR Cycling (40 cycles) 1.) 95° C., 30 sec. 2.) 55° C., 30sec. 3.) 72° C., 1 min; Melt Curve (Dissociation Stage). The primersequences for the genes analyzed are

GAPDH Forward (SEQ ID NO: 30) 5′-GGCCTCCAAGGAGTAAGACC; GAPDH Reverse(SEQ ID NO: 31) 5′-AGGGGTCTACATGGCAACTG; QSOX1-S Forward (SEQ ID NO: 32)5′-TGGTCTAGCCACAACAGGGTCAAT; QSOX1-S Reverse (SEQ ID NO: 33)5′-TGTGGCAGGCAGAACAAAGTTCAC; QSOX1-L Forward (SEQ ID NO: 34)5′-TTGCTCCTT GTCTGGCCTAGAAGT; QSOX1-L Reverse (SEQ ID NO: 35)5′-TGTGTCAAAGGAGCTCTCTCTGTCCT; MMP-2 Forward (SEQ ID NO: 36)5′-TTGACGGTAAGGACGGACTC; MMP-2 Reverse (SEQ ID NO: 37)5′-ACTTGCAGTACTCCCCATCG; MMP-9 Forward (SEQ ID NO: 38)5′-TTGACAGCGACAAGAAGTGG; and MMP-9 Reverse (SEQ ID NO: 39)5′-CCCTCAGTGAAGCGGTACAT.Each reaction was performed in triplicate with the data representing theaverages of one experiment.

In the shRNA experiment, expression of MMPs was normalized to thenon-targeted GAPDH to determine ΔCq. ΔCq replicates were thenexponentially transformed to the ΔCq expression after which they wereaveraged±standard deviation. The average was then normalized to theexpression of the shScramble control to obtain the ΔΔCq expression.Significance was determined using the Student's two-tailed t-Test.

Matrigel™ Invasion Assay

Invasion assays were performed using BD BioCoat™ BD Matrigel™ Invasionchambers with 8.0 μm pore size polyethylene terephthalate (PET) membraneinserts in 24-well format. The assay was performed according to themanufacturers' instructions (BD Bioscience). 4×10⁴ cells/well wereseeded into the inner Matrigel™ chamber in serum free DMEM. The outerchamber contained 10% FBS in DMEM. BxPC3 and Pane-1 cells were incubatedfor 24 hours at 37° C., 5% CO₂. Cells that invaded through the Matrigel™and migrated through the pores onto the bottom of the insert were fixedin 100% methanol and then stained in hematoxylin (Invitrogen). The totalnumber of invading cells were determined by counting the cells on theunderside of the insert from three wells (6 fields per insert) at 10×,20× and 40× magnification and the extent of invasion was expressed asthe average±standard deviation. Significance was determined using theStudent's two-tailed t-Test. Results presented are from one of threeindependent experiments.

Results for Example 1 Detection of QSOX1 by Immunohistochemistry andWestern Blot

To begin to determine the frequency of expression of QSOX1 in human PDA,QSOX1 expression was assessed in 4 different pancreatic tumor celllines, an immortal non-tumorigenic cell line, HPDE6, 37 tumor tissuesections from patients with PDA, and tumor and adjacent normal tissuefrom two patients, 1016 and 1010 (FIGS. 1B, C, and D). 29 of 37 tumortissues were positive for QSOX1 expression, suggesting it is a commonlyover-expressed protein. To determine which splice variant was moreprevalent in our IHC images we analyzed tumor as well as adjacent normaltissue from 2 patients by western blot (FIG. 1C). Our results revealedthat QSOX1-S is the dominant splice variant expressed also corroboratingour IHC results that revealed an increase in QSOX1 expression in tumorsamples. While our adjacent normal samples indicate a high level ofQSOX1 expression, it is hard to determine if there was any tumor tissuecontaminating our normal sample, which would account the increase inQSOX1 expression. Western blotting analysis shows that 4 pancreatictumor cell lines, BxPC3, Panc-1, Capan1 and CFPac1 strongly expressQSOX1-S and weakly express the longer splice variant, QSOX1-L. HPDE6, animmortal, non-tumorigenic pancreas epithelial cell line, shows weakexpression of QSOX1-S and no expression of QSOX1-L (FIG. 1D).

The results of this experiment begin to provide some information aboutthe frequency and distribution of QSOX1 expression. First, QSOX1 appearsto be a commonly over-expressed protein in PDA (FIG. 1B-C). Second,QSOX1 protein expression in adjacent normal 1016, 1010, and HPDE6, anon-tumorigenic pancreatic duct cell line, is much weaker than it is inthe patient tumor samples and four malignant pancreatic tumor celllines. This may suggest that QSOX1 provides some advantage to malignantcells that non-malignant cells do not require.

QSOX1 Promotes Tumor Cell Proliferation

To examine the advantage that QSOX1 provides to tumor cells we inhibitedQSOX1 expression in BxPC3 and Panc-1 cells using 3 shRNA constructs:sh742, sh528 and sh616. shScrambled was generously provided by Dr.Joshua LaBaer. Lentiviruses containing each shRNA were generated asdescribed in “Methods.” BxPC3 and Panc-1 cells were transduced with eachsh-lentivirus (shQSOX1) to evaluate the effects of QSOX1 knockdown ontumor cell growth. To demonstrate that the shQSOX1 are active in bothcell lines, FIG. 2A-B shows reduced protein expression of both isoformsof QSOX1 in BxPC3 and Panc-1 tumor cell lines compared to scrambledshRNA in western blot analysis. This experiment demonstrates that sh742,sh528 and sh616 knock down of QSOX1-S expression in BxPC3 cells was 56%,40% and 28%, respectively; for Panc-1 cells the knock down was 64%, 46%and 18%, respectively (FIG. 2A-B).

ShQSOX1-transduced BxPC3 and Panc-1 cells exhibited a decrease in cellgrowth compared to shScrambled controls in an MTT assay (FIG. 2C) and byTrypan blue viable cell dye (not shown). We seeded an equal number ofshScramble, sh742, sh528 and sh616 cells in 96 well plates andquantified the proliferation rate by measuring mitochondrial metabolismon days 1, 2 and 5. While on day 1 there was no change, day 2 presenteda minor decrease in cell growth by day 5 BxPC3 sh742, sh528 and sh616showed a 65%, 60% and 37% decrease, while in Panc-1 sh742, sh528 andsh616 there was a 84%, 88% and 61% decrease. Live cell counts usingTrypan blue confirmed the MTT assay (not shown).

Cell Cycle and Apoptosis Analysis

Previous work has correlated QSOX1 expression with the quiescent stage,G₀, of the cell cycle (10), leading us to hypothesize if theshQSOX1-mediate decrease in cell proliferation was the result ofabnormal regulation of the cell cycle or an increase in apoptosis. Toaddress this hypothesis, propidium iodide (PI) was used in flowcytometry to evaluate the effects of shQSOX1 on cell cycle. Our resultsindicate that suppression of QSOX1 expression did modulate cell cycle inboth BxPC3 and Panc-1 compared to our untreated and scrambled control(data not shown). The results show that the reduced expression of QSOX1on cell cycle could be cell dependent. BxPC3 showed an increase in G₁and a significant decrease in S, while Panc-1 cells showed a significantdecrease in G₁ but no changes in S.

We further evaluated if the decrease in cellular proliferation mediatedby shQSOX1 was due to an increase in apoptotic cell death. To assessapoptosis, BxPC3 and Panc-1 cells transduced with shScramble, sh742,sh528 and sh616 were stained with annexin-V and PI. Compared tountreated and shScramble a consistent increase of 2-8% in early and lateapoptosis (Annexin-V single and double positive) was observed for eachof the shQSOX1 constructs in BxPC3 and Panc-1 cells. Indicating that thereduced expression of QSOX1 does lead to cell death but does notentirely account for the dramatic decrease in cellular proliferation.This data also agrees with our viable cell count revealing a largelynonsignificant decrease in shQSOX1 viable cells compared to untreatedand shScramble controls.

Role of QSOX1 in Tumor Cell Invasion

For a tumor cell to invade other tissues as part of the metastaticprocess, the cell must first degrade basement membrane components suchas laminin, collagen and fibronectin before it can migrate into theblood stream and re-establish itself in a distant organ (3). To evaluatewhether over-expression of QSOX1 in BxPC3 and/or Pane-1 cells plays arole in metastasis we performed invasion assays over an 18-hour period.Untreated, shScramble, sh742, sh528 and sh616-transduced cells wereplated in serum-free medium on Matrigel™-coated, 8 um pore inserts.Inserts were placed into wells containing 10% FBS in DMEM. After 18hours of incubation, tumor cells that had degraded Matrigel™ andmigrated through 8 um pores onto the underside of the insert werecounted (FIG. 4A-B). Our results clearly demonstrate that knockdown ofQSOX1 expression in tumor cells leads to a dramatic decrease in thenumber of pancreatic tumor cells that degrade Matrigel™ and migratethrough the insert into nutrient rich media.

Mechanism of Invasion

Since knock-down of QSOX1 protein expression in pancreatic tumor cellslines decreases invasion through Matrigel™, it was important todetermine the mechanism of inhibition of the invasive process. MMP-2 and-9 are key contributors of invasion and metastasis in pancreatic cancer(2). Both, pro-MMP-2 and -9 mRNA and protein levels are elevated inpancreatic tumors, and activated MMP-2 (a-MMP2) appears to be keycontributors of metastasis in PDA (2, 22). Because QSOX1 has beensuggested to be secreted into the extracellular matrix where MMPs arethought to be activated, we hypothesized that QSOX1 might help activateMMP-2 and -9 proteins. Untreated BxPC3 and Panc-1 cells, as well astransduced shScramble, sh742, sh528 and sh616 were incubated for 18-24hours in serum free media after which supernatants were collected andsubjected to gelatin-SDS-PAGE. Gelatin zymography was performed todetermine if QSOX1 plays a role in secretion and/or activation of MMPs.

Our first observation from this experiment is that BxPC3 and Panc-1 havevery different zymographic profiles. BxPC3 supernatants contain MMP-9homodimer (130 kDa), a large amount of proteolytically active pro-MMP-9(92 kDa) with lesser concentrations of pro-MMP-2 (72 kDa) and a-MMP-2(66 kDa). Panc-1 supernatants contain less prominent MMP-9 homodimer,pro-MMP-9 (92 kDa) and a large amount of proteolytically activepro-MMP-2 (72 kDa), unlike BxPC3 cells.

Supernatants from BxPC3 cells transduced with sh742, sh528 and sh616showed a 65%, 47% and 10% decrease, respectfully, in pro-MMP9 comparedto shScramble (FIG. 5A). Supernatants from Panc-1 cells tranduced withsh742, sh528 and sh616 showed a 70%, 56% and 15% decrease, respectfully,in pro-MMP-2. Thus, decreases in the proteolytic activity of MMP-2 and-9, using gelatin as a substrate, provide a mechanism for theshQSOX1-mediated suppression of invasion through Matrigel™.

To confirm our gelatin zymography results we used western blot analysisof BxPC3 and Panc-1 serum free conditioned media to probe for MMP-2 and-9 (FIG. 5C). While our results indicate a slight decrease in MMP-2 and-9 (between 1 and 10% decrease using densitometry analysis) in BxPC3 andPanc-1 shQSOX1 treated cells it is nowhere near the level shown usinggelatin zymography. This could be explained as a difference between afunctional assay, gelatin zymography, and a purely quantitative assaysuch as western blot.

To extend our hypothesis that QSOX1 is influencing MMPspost-translationally, we performed quantitative real time PCR (QRTPCR)on MMP-2 and MMP-9 comparing the transcripts from shQSOX1 transducedcell lines with shScrambled. FIG. 5 demonstrates that MMP-2 and -9 RNAincreased in the shQSOX1 transduced cells compared to control cells.This result adds confidence to our hypothesis that QSOX1 does nottranscriptionally suppress MMP production, rather itpost-translationally suppresses MMP activity. It also diminishes thepossibility that shQSOX1 RNAs are suppressing MMP transcription due tooff-target effects.

Discussion for Example 1

The mortality rate for patients diagnosed with pancreatic cancer hasremained stagnant for the last five decades despite advanced surgicalprocedures and improvements in chemotherapeutics (23). Because mostpatients present with advanced metastatic disease, it is critical tounderstand the properties of invasive pancreatic tumors. Discovery andsubsequent study of factors that contribute to tumor cell invasionprovide an opportunity to develop therapeutics that could be used aloneor in combination with other anti-neoplastic agents. Prior to our report(4), it was not previously known that QSOX1 was over-expressed inpancreatic tumors. The results presented in FIG. 1 suggest that QSOX1 isa commonly over-expressed protein in PDA, making it a potential target.To extend those initial findings we began to investigate why pancreatictumors over-express QSOX1, and mechanistically, what advantage itaffords tumors.

Tumor cells in which QSOX1 protein expression was suppressed by shQSOX1grew more slowly than the shScrambled and untreated controls as measuredby an MTT assay, while the results with our strongest shQSOX1, sh742,show a greater that 50% decrease in cell growth in both BxPC3 and Panc-1cells (FIG. 2C). Our attempt to try and explain the decrease inproliferation as a result of abnormal cell cycle regulation or anincrease in apoptosis do not show a similar level of change that cansolely explain our MTT results (FIG. 3, S2). Contrary to previousstatements implicating QSOX1 as a cell cycle regulator (24), our resultssuggest that while the loss of QSOX1 in Panc-1 cells shows a consistentdecrease in G₁, there is no where near that effect when we analyzedBxPC3 cells suggesting that the role of QSOX1 could be cell type andtumor stage dependent, as a result of the different substrates available(S2). Our results likely conflict because we assessed the effect ofQSOX1 on pancreatic tumor cell growth, not fibroblasts where QSOX1 wasinitially described (5, 24).

The same statement can be made in regards to the loss of QSOX1 directlyaffecting apoptosis. While our strongest knock-down, sh742, does show atits greatest an 8% increase in annexin V/propidium iodide doublepositive cells it is not enough to explain the dramatic decrease incellular proliferation (FIG. 3). There are numerous proteins within thecell that assist in disulfide bond formation that may compensate for theloss of QSOX1 such as protein disulfide isomerase (PDI), thioredoxin,glutathione and members of the Erv family of sulfhydryl oxidases (25).There are no known preferred substrates of QSOX1 although speculationbased on the function of QSOX1 as well as the known substrates thatcorrespond to QSOX1 functional domains, leads us to believe that thereare a broad spectrum of possible substrates and therefore the role thatQSOX1 plays in tumor cell progression would most likely be influenced bythe substrates with the greatest affinity for QSOX1. Compensation bythese other oxidases could help explain why the loss of QSOX1 does notlead to significant alterations in the cell cycle and apoptosis. It isalso possible that suppression of QSOX1 activity does not induceapoptosis, but results in other phenomena such as anoikis or autophagy(26). We may investigate these possibilities in future studies.

Another hallmark of cancer is invasion. Since suppression of QSOX1 didnot appear to play a major role in tumor cell growth, we hypothesizedthat the over-expression of QSOX1 in pancreatic tumor cells maycontribute to their ability to degrade basement membranes, leading to aninvasive and metastatic phenotype. We discovered that suppression ofQSOX1 protein resulted in a dramatic reduction in the ability of bothBxPC3 and Panc-1 pancreatic tumor cells to invade through Matrigel™ invitro (FIG. 4). It is clear through these results that there are cleardifferences between BxPC3 and Panc-1 ability to degrade basementmembrane components and invade. This could be due to a myriad of factorssuch as the proteases secreted, the stage of the tumor and geneticdifferences between the two cells lines (27). To determine if thisreasoning was correct, we performed gelatin zymography as a way toanalyze the matrix metalloproteinase activity.

As a sulfhydryl oxidase, it is unlikely that QSOX1 would directlydegrade basement membrane components. Therefore, we hypothesized thatMMPs serve as a substrate of QSOX1 while the MMPs are folding andundergoing activation as they are secreted from tumor cells. If true,suppression of QSOX1 would lead to a decrease in MMP functionalactivity, though not necessarily the amount of MMPs produced orsecreted. Although the MMP profiles of BxPC3 and Panc-1 cells differ asseen in FIGS. 5A and B, we found that suppression of QSOX1 leads to adecrease in pro-MMP-2 and -9 activity. MMPs are zinc-dependentproteolytic enzymes that degrade ECM components (22). There are 23 knownhuman MMPs as well as 4 known tissue inhibitors of MMPs (TIMP) that aidin regulating the expression and activation of these proteolytic enzymes(22). The expression patterns of MMPs are variable depending on tumortype, and even individual cell line.

In pancreatic cancer the majority of MMPs are secreted in their inactiveform and activated extracellularly (28). Activation of MMPs occurseither through the release of a covalent Cys⁷³-Zn²⁺ bond (“CysteineSwitch”) or through cleavage and activation by plasmin, serineproteases, and other MMPs or TIMPs (21, 28). MMP-2 and -9 have beenfound to play an important role in pancreatic cancer progression with93% of tumors expressing MMP-2 compared to normal tissue (28). Whilereports implicating MMP-9 in the progression of pancreatic cancer arelimited, Tian reported the proteomic identification of MMP-9 inpancreatic juice from patients with pancreatic ductal adenocarcinoma(29). Pryczynicz et al. also found a relationship between MMP-9expression and lymph node metastases (30). Numerous reports implicateMMP-2 as a prominent protease responsible for pancreatic tumormetastasis (2, 22, 28).

One of the benefits of gelatin zymography is that it a.) provides afunctional measure of the activities of MMPs able to degrade gelatin andb.) differentiates each precursor and active MMP by molecular weight(21, 31). A limitation of the zymography shown here is that it islimited to MMPs whose substrate is gelatin. It is possible that QSOX1 isinvolved in activation of other MMPs with different substrates. Thiswill be investigated in future work.

Following up on our initial hypothesis regarding MMP activation by QSOX1we performed a western blot analysis on the same serum free conditionedmedia that was used to perform gelatin zymography. Our result revealedthat the overall levels of secreted MMP-2 and -9 are nearly equal amongthe untreated, shScramble and shQSOX1 treated samples leading us tofurther hypothesize that QSOX1 is involved in the proper folding of MMPsbefore they are secreted and that the loss of QSOX1 leads toproteolytically inactive MMPs as shown in FIGS. 5A, B and C. To furtherconfirm that what we are observing is a post-translational event weperformed qPCR on BxPC3 and Panc-1 shQSOX1 treated cells (FIG. 5D-E).Our observation was surprising in that we are able to show that there isan overall increase in the transcription of MMP-2 and -9. This resultled us to hypothesize that the cell is transcriptionally attempting tocompensate for the proteolytically inactive MMPs through an as yetundetermined mechanism.

QSOX1 was previously reported by our group to be over-expressed inpatients diagnosed with pancreatic cancer (4), and that a peptide fromthe QSOX1 parent protein is present in plasma from patients with PDA. Inthe present study we demonstrated for the first time that expression ofQSOX1 in pancreatic tumor cells directly contributes to an invasive andpotentially metastatic phenotype through the activation of MMP-2 and -9through an as yet undetermined mechanism. It is not known if QSOX1 issolely responsible for the proper folding of MMPs intracellularly, or ifit cooperates with protein disulfide isomerase while MMPs are folding inthe ER and golgi. Since MMPs are secreted extracellularly where they mayundergo autoactivation or cleavage with proteases such as plasmin, it ispossible that QSOX1-S activates them in the extracellular environment.

At this point, the post-translational mechanism by which QSOX1 activatesMMPs is not clear. Our results indicate that MMP-2 and -9 RNA increasedin shQSOX1 transduced cells. We expected no difference in MMP levels,but an increase might suggest that the cells are attempting tocompensate for the lack of MMP activity through a feedback loop (32,33). Although we hypothesize that QSOX1 may activate MMPs directly byinvolvement in the cysteine switch activation mechanism (21, 28), ROSproduced by QSOX1 may be indirectly activating MMPs, as MMP activationhas been reported to depend on an oxidative environment (32, 33).

Our results underscore the need to further understand the role thatQSOX1 plays in tumor and normal cells, and how at the molecular level,it activates MMPs. This information will be useful during development ofinhibitors of QSOX1 that may work upstream of individual MMPs.

REFERENCES FOR EXAMPLE 1

-   1. Bardeesy N, DePinho R A. Pancreatic cancer biology and genetics.    Nature Publishing Group 2002; 12:897-909.-   2. Koshiba T, Hosotani R, Wada M, Miyamoto Y, Fujimoto K, Lee J U,    et al.-   Involvement of matrix metalloproteinase-2 activity in invasion and    metastasis of pancreatic carcinoma. Cancer; 1998. p. 642-50.-   3. Strimpakos A, Saif M W, Syrigos K N. Pancreatic cancer: from    molecular pathogenesis to targeted therapy. Cancer Metastasis Rev    2008; 3:495-522.-   4. Antwi K, Hostetter G, Demeure M J, Katchman B A, Decker G A, Ruiz    Y, et al. Analysis of the plasma peptidome from pancreas cancer    patients connects a peptide in plasma to overexpression of the    parent protein in tumors. J Proteome Res 2009; 10:4722-31.-   5. Coppock D L, Cina-Poppe D, Gilleran S. The quiescin Q6 gene    (QSCN6) is a fusion of two ancient gene families: thioredoxin and    ERV1. Genomics 1998; 3:460-8.-   6. Coppock D. Regulation of the Quiescence-Induced Genes: Quiescin    Q6, Decorin, and Ribosomal Protein S29. Biochemical and Biophysical    Research Communications 2000; 2:604-10.-   7. Heckler E J, Alon A, Fass D, Thorpe C. Human quiescin-sulfhydryl    oxidase, QSOX1: probing internal redox steps by mutagenesis.    Biochemistry 2008; 17:4955-63.-   8. Coppock D L, Thorpe C. Multidomain flavin-dependent sulfhydryl    oxidases. Antioxid Redox Sign 2006; 3-4:300-11.-   9. Hoober K L, Thorpe C. Flavin-dependent sulfhydryl oxidases in    protein disulfide bond formation. Methods Enzymol 200230-4.-   10. Thorpe C, Hoober K L, Raje S, Glynn N M, Burnside J, Turi G K,    et al. Sulfhydryl oxidases: emerging catalysts of protein disulfide    bond formation in eukaryotes. Arch Biochem Biophys 2002; 1:1-12.-   11. Vitu E, Bentzur M, Lisowsky T, Kaiser C A, Fass D. Gain of    function in an ERV/ALR sulfhydryl oxidase by molecular engineering    of the shuttle disulfide. J Mol Biol 2006; 1:89-101.-   12. Alon A, Heckler E J, Thorpe C, Fass D. QSOX contains a    pseudo-dimer of functional and degenerate sulfhydryl oxidase    domains. FEBS Lett 2010; 8:1521-5.-   13. Hoober K L, Joneja B, White H B, 3rd, Thorpe C. A sulfhydryl    oxidase from chicken egg white. J Biol Chem 1996; 48:30510-6.-   14. Hoober K L, Glynn N M, Burnside J, Coppock D L, Thorpe C.    Homology between egg white sulfhydryl oxidase and quiescin Q6    defines a new class of flavin-linked sulfhydryl oxidases. J Biol    Chem 1999; 45:31759-62.-   15. Morel C, Adami P, Musard J, Duval D, Radom J, Jouvenot M.    Involvement of sulfhydryl oxidase QSOX1 in the protection of cells    against oxidative stress-induced apoptosis. Experimental Cell    Research 2007; 19:3971-82.-   16. Song H, Zhang B, Watson M A, Humphrey P A, Lim H, Milbrandt J.    Loss of Nkx3.1 leads to the activation of discrete downstream target    genes during prostate tumorigenesis. Oncogene; 2009. p. 3307-19.-   17. Ouyang X, DeWeese T L, Nelson W G, Abate-Shen C.    Loss-of-function of Nkx3.1 promotes increased oxidative damage in    prostate carcinogenesis. Cancer Research; 2005. p. 6773-9.-   18. Bowen C, Bubendorf L, Voeller H J, Slack R, Willi N, Sauter G,    et al. Loss of NKX3.1 expression in human prostate cancers    correlates with tumor progression. Cancer Res 2000; 21:6111-5.-   19. Ouyang H, Mou L, Luk C, Liu N, Karaskova J, Squire J, et al.    Immortal human pancreatic duct epithelial cell lines with near    normal genotype and phenotype. Am J Pathol 2000; 5:1623-31.-   20. Zuo D, Mohr S E, Hu Y, Taycher E, Rolfs A, Kramer J, et al.    PlasmID: a centralized repository for plasmid clone information and    distribution. Nucleic Acids Res 2007; Database issue:D680-4.-   21. Snoek-van Beurden PAM, Von den Hoff J W. Zymographic techniques    for the analysis of matrix metalloproteinases and their inhibitors.    BioTechniques 2005; 1:73-83.-   22. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases:    regulators of the tumor microenvironment. Cell 2010; 1:52-67.-   23. Koorstra J B, Feldmann G, Habbe N, Maitra A. Morphogenesis of    pancreatic cancer: role of pancreatic intraepithelial neoplasia    (PanINs). Langenbecks Arch Surg 2008; 4:561-70.-   24. Coppock D L, Kopman C, Scandalis S, Gilleran S. Preferential    gene expression in quiescent human lung fibroblasts. Cell Growth    Differ 1993; 6:483-93.-   25. Fass D. The Ery family of sulfhydryl oxidases. Biochim Biophys    Acta 2008; 4:557-66.-   26. Evan G I, Vousden K H. Proliferation, cell cycle and apoptosis    in cancer. Nature 2001; 6835:342-8.-   27. Firpo M A, Deer E L, Gonzalez-Hernandez J, Coursen J D, Shea J    E, Ngatia J, et al. Phenotype and Genotype of Pancreatic Cancer Cell    Lines. Pancreas 2010; 4:425-35.-   28. Bloomston M, Zervos E E, Rosemurgy A S. Matrix    metalloproteinases and their role in pancreatic cancer: a review of    preclinical studies and clinical trials. Ann Surg Oncol 2002;    7:668-74.-   29. Tian M, Cui Y Z, Song G H, Zong M J, Zhou X Y, Chen Y, et al.    Proteomic analysis identifies MMP-9, DJ-1 and A1B G as overexpressed    proteins in pancreatic juice from pancreatic ductal adenocarcinoma    patients. BMC Cancer 2008241.-   30. Pryczynicz A, Guzinska-Ustymowicz K, Dymicka-Piekarska V,    Czyzewska J, Kemona A. Expression of matrix metalloproteinase 9 in    pancreatic ductal carcinoma is associated with tumor metastasis    formation. Folia Histochem Cytobiol 2007; 1:37-40.-   31. Kupai K, Szucs G, Cseh S, Hajdu I, Csonka C, Csont T, et al.    Matrix metalloproteinase activity assays: Importance of zymography.    Journal of Pharmacological and Toxicological Methods 2010; 2:205-9.-   32. Wu W S. The signaling mechanism of ROS in tumor progression.    Cancer Metast Rev 2006; 4:695-705.-   33. Kar S, Subbaram S, Carrico P M, Melendez J A. Redox-control of    matrix metalloproteinase-1: a critical link between free radicals,    matrix remodeling and degenerative disease. Respir Physiol Neurobiol    2010; 3:299-306.

Example 2

We designed two short hairpin RNAs (shRNA1 and shRHA2) to knock-downQSOX1 protein expression in tumor cells.

shRNA1 - Targeting QSOX1 sequence begins at the 970^(th) base pair -(SEQ ID NO: 17) ATCTACATGGCTGACCTGGAA shRNA2 - Targeting QSOX1 sequencebegins at the 1207^(th) base pair - (SEQ ID NO: 18)AGGAAAGAGGGTGCCGTTCTT

QSOX1 shRNA1 hybridizes with nucleotides 970-989 of the QSOX1transcript. QSOX1 shRNA2 hybridizes to nucleotides 1207-1226 of thetranscript. Both shRNAs, independently and together suppressed theproduction of QSOX1 protein (data not shown) and cell proliferation.Upon cloning the QSOX1 shRNA into a eukaryotic expression vector (pCS2mammalian overexpression vector with a CMV promoter) and using it totransfect a pancreatic tumor cell line (BxPC-3), a 30-40% decrease incell viability was observed, over a 4 to 6 day period, compared tountreated and scrambled shRNA controls. (FIG. 6) Using the sametransfection protocol in a separate experiment, we made an additionalobservation that QSOX1 shRNA-transfected BxPC-3 cells were inhibitedfrom invading through a Matrigel™ basement membrane composed ofcollagen, laminin and fibronectin approximately 70% when both QSOX1shRNAs were combined (FIG. 7).

A summary of the data obtained from these studies is shown in the tablebelow:

Invasion of tumor % Decrease in cells through a QSOX1 protein basementViability/mitochondrial Sample expression membrane respiration shRNA1 3763 39 shRNA2 10 69 48 shRNA1/2 41 77 50

Values in table represent % decreases in protein expression or activitycompared to scrambled shRNA in the same CMV driven pCS2 mammalianoverexpression vector, Knockdown is transient in this pCS2 vector.

Example 3

MIAPaCa2 and Panc-1 pancreatic tumor cells were transduced with shRNAthat specifically knocked down QSOX1 protein in the tumor cells. TheshRNAs used to knockdown QSOX1 in tumor cells were

QSOX1 sh742 (SEQ ID NO: 26) (5′-CCGG GCCAATGTGGTGAGAAAGTTT CTCGAGAAACTTTCTCACCACATTGGCTTTTTG-3′) and shRNA QSOX1 sh528 (SEQ ID NO: 21)5′-CCGGACAATGAAGAAGCCTTT-3′ (sense),Nude mice Human pancreatic tumor cells (MIAPaCa2) were transduced with alentivirus encoding shQSOX1 (sh528 and sh742) and shScramble (control).One million MIAPaCa2 cells were mixed with Matrigel™ and used toinoculate nude mice (5 mice/group) on day 0. After day 12, tumor growthwas measured every 3 days (x-axis) and reported as “Tumor volume” on theY-axis. “Untreated” indicates that tumor cells were not transduced. (seeFIGS. 8 and 9). This in vivo experiment thus validates QSOX1 as apotential target for anti-neoplastic drugs. Any other type of inhibitorof QSOX1 expression or function would have a similar effect on tumorcell growth.

Example 4

In this present study, we evaluated QSOX1 protein expression in breastadenocarcinoma cell lines MCF7, BT474 and BT549 and in a breast tumortissue microarray. Using short hairpin RNA (shRNA) specific for QSOX1-Sand -L, we assessed the effects of QSOX1 knockdown on cell growth, cellcycle, apoptosis, invasion and matrix metalloproteinase activity. Theloss of QSOX1 significantly affected tumor cell proliferation anddramatically suppressed tumor cell invasion through Matrigel™ Theaddition of exogenous recombinant human QSOX1 (rhQSOX1) rescued theinvasive capabilities of MCF7, BT474 and BT549 validating thepro-invasive function of QSOX1. We further report the mechanism ofQSOX1-mediated invasion in vitro is due in part, to elevated MMP-9activity.

Expression of QSOX1 Correlates with Poor Prognosis in Patients withLuminal B Breast Cancer

Bioinformatic analysis of QSOX1 transcript expression was assessed usingdata from the Gene expression based Outcome for Breast cancer Onlinealgorithm (GOBO) [17]. GOBO is a web based analysis tool that utilizesAffymetrics gene expression data curated from 1,881 breast cancerpatients with associated stage, grade, nodal status and intrinsicmolecular classification based on the paradigm first reported by thePerou Laboratory [18]. Expression of QSOX1 was significantly higher inER+ tumors compared to ER− (P<0.00001), with the highest expressionobserved in Luminal A, Luminal B and Normal-like subtypes (FIG. 12a, b). The lowest QSOX1 transcript expression was observed in HER2-enrichedand basal tumors. Using the GOBO tool, we performed a series of KaplanMeier analyses to determine whether QSOX1 expression is associated withrelapse free survival (RFS) and overall survival (OS) (FIG. 12c, d ).While elevated QSOX1 expression is not associated with survival whenconsidering all breast tumor subtypes together, it is highly associatedwith poor RFS (P=0.00062) and OS (P=0.00031) in Luminal B tumors (FIG.12c, d ). The expression of QSOX1 correlates with increasing tumor gradeas well as poor overall survival in patients diagnosed with grade 2(P=0.04242) and grade 3 (P=0.07095) breast tumors. Elevated QSOX1 wasalso associated with reduced OS in Luminal A tumors and is a predictorof poor OS for patients who did not receive systemic treatment (data notshown).

Evaluation of QSOX1 Expression by Immunohistochemistry

Results from the GOBO transcript expression analysis fueledinvestigation of QSOX1 at the protein level in breast tumors. A breasttumor tissue microarray composed of breast tumors from over 150different patients was stained with a rabbit anti-QSOX1 polyclonalantibody and scored by a board certified pathologist (ITO). FIG. 13bshows no expression of QSOX1 in normal breast tissue. FIGS. 13c-frepresent a pattern of increasing QSOX1 expression observed in the TMAin grade 1, grade 2 and grade 3 invasive ductal carcinomas and a grade 3invasive lobular carcinoma. Statistical evaluation of QSOX1 expressionby immunohistochemistry (IHC) demonstrated an association with ER+tumors, and a strong association with high Ki-67 expression in patientswith a high QSOX1 IHC score (FIG. 13a , Table 1). There was norelationship observed for QSOX1 expression in HER2+ tumors orcytokeratin markers (CK 5/6) positive tumors. These data are consistentwith the correlation observed in the GOBO data. Interestingly, highergrade tumors were associated with a higher QSOX1 IHC score (FIG. 13a ,Table 1). Conversely, lower QSOX1 protein expression is significantlyassociated with lower grade tumors. This is consistent with anassociation between QSOX1 expression and more aggressive ER+ tumors.

TABLE 1 Statistical assessment of QSOX1 protein expression withmolecular subtypes of breast cancer IHC Score High No QSOX1 Low QSOX1Intermediate QSOX1 staining staining QSOX1 staining staining (n = 17) %(n = 47) % (n = 24) % (n = 65) % P-value Grade *0.0003 1 53.3 42.2 2510.8 2 33.3 33.3 41.7 32.3 3 13.3 24.4 33.3 56.9 ER *0.0013 ER+ 80 89.173.9 55.4 ER− 20 10.9 26.1 44.6 HER2 0.0811 HER2+ 11.8 6.4 29.2 14.1HER2− 88.2 93.6 70.8 85.9 CK5/6 0.0733 CK5/6− 100 95.7 87.5 83.1 CK5/6+0 4.3 12.5 17 KI-67 *0.0011 Low 33.3 33.3 41.1 18.5 Intermediate 44.445.5 17.7 16.7 High 22.3 21.2 41.2 64.8 ER & HER2 *0.0016 ER− HER2− 13.38.7 8.7 35.9 Others 86.7 91.3 91.3 64.1 ER, HER2 and 0.0923 CK5/6 ER−HER2−, 0 4.3 4.2 15.4 CK5/6: 1/2/3 Others 100 95.7 95.8 84.6

Evaluation of QSOX1 Expression by Western Blot

QSOX1 expression in human breast adenocarcinoma was assessed in sixdifferent breast tumor cell lines, and a transformed non-tumorigenicbreast cell line, MCF10A [19, 20]. Similar to our studies in pancreaticcancer, the short form of QSOX1 is expressed as the predominant splicevariant in each cell line examined (FIG. 14a ). Consistent with the GOBOand IHC expression data, we found that the expression of QSOX1-S proteinwas more highly expressed in luminal-like cell lines MCF7 (ER+),MDA-MB-453 (ER−), ZR 75 (ER+) and BT474 (ER+) compared to basal-likeBT549 and MDA-MB-231 cell lines. Interestingly, QSOX1 was most weaklyexpressed in transformed normal MCF10A cells, which do not form tumorsin immunodeficient animals.

Expression of QSOX 1 in Tumor Cells Promotes Cellular Proliferation

To begin to assess the mechanistic role that QSOX1 plays in tumor cellswe stably knocked-down QSOX1 expression in MCF7, BT549 and BT474 cellsusing two lentiviral shRNA constructs, sh742 and sh528 (data not shown).QSOX1 protein expression was assessed following stable knock-downrelative to isogenic parental cell lines by western blotting.Densitometry of the QSOX1 protein relative to alpha-tubulin expressionindicates that sh742 and sh528 resulted in a knock-down of QSOX1-Sexpression in MCF7 cells by 85% and 82%, respectively. In BT549 cellsthe knock-down was 65% and 77%, and for BT474 cells by 40% and 36%,respectively.

The growth rates of shQSOX1-transduced MCF7, BT549 and BT474 cells werethen evaluated compared to isogenic controls. An equal number ofuntransduced (parental), shScramble, sh742 and sh528 cells were seededin 96 well plates and assayed for proliferation over 5 days using theMTT assay. ShQSOX1-transduced MCF7, BT549 and BT474 cells displayed adecrease in cell growth compared to shScrambled and parental controls.In MCF7 cells, sh742 and sh528 showed a 66% decrease in cell growth,while sh742 and sh528 suppressed growth of BT549 by 78% and 69%,respectively, and sh742 and sh528 suppressed growth of BT474 by 52% and29%, respectively by day 5. We confirmed our MTT results by performingTrypan blue staining over 5 days using the same incubation conditions asin the MTT assay. These results suggest that QSOX1 helps drive tumorcell growth.

Cell Cycle, Apoptosis and Autophagy Analysis

We hypothesized that a shQSOX1-mediated decrease in cell proliferationcould be the result of abnormal regulation of the cell cycle, anincrease in apoptosis or the result of autophagosome formation. Toaddress this, propidium iodide (PI) was used in flow cytometry toevaluate the effects of shQSOX1 on cell cycle. In MCF7 cells, bothshQSOX1 RNAs showed a slight decrease in G₁ and an increase (11-12%) inS phase, while in BT474 cells both shQSOX1 RNAs showed a slight 12%increase in G₁ and a 26% decrease in S phase but neither shQSOX1 RNAsequence had any effect in BT549 cells compared to untreated andshScramble controls (data not shown).

Next we determined if the decrease in cellular proliferation was due toan increase in apoptosis or autophagy. To assess apoptosis, we analyzedMCF7 and BT474 transduced cells for Annexin V/PI [18]. We subsequentlyprobed MCF7 and BT549 transduced cells for LC3, a protein that isnecessary for auotphagosome formation [19]. If the expression of QSOX1prevented cellular apoptosis or autophagy we would expect to see anincrease in expression of Annexin V and LC3 in shQSOX1 transduced cells,but we did not observe any statistically significant increases inAnnexin V positive cells (data not shown). This correlates with ourprevious results in pancreas cancer that the suppression of QSOX1 doesnot lead to cell death or autophagy.

Suppression of QSOX1 Expression Inhibits Tumor Cell Invasion

The process of tumor cell invasion involves the degradation of basementmembrane (BM) components such as laminin, collagen and fibronectinbefore a tumor cell is able to invade other tissues [20]. We performed amodified Boyden chamber assay using Matrigel™-coated inserts in whichtumor cells must degrade the Matrigel™ and migrate through a membranewith 8 um pores to gain access to nutrient rich media. Sh742 andsh528-transduced MCF-7, BT549 and BT474 tumor cells were added toMatrigel™-coated, 8 um pore inserts in serum-free medium. After 72(MCF7) and 48 (BT549 and BT474) hours of incubation, tumor cells thatwere able to degrade Matrigel™ and migrate through 8 um pores onto theunderside of the insert were counted (FIGS. 10a, b and c ). Our resultsdemonstrate that knockdown of QSOX1 expression in MCF7 leads to a 65%and 71% reduction in invasion of sh742 and sh528 transduced tumor cells,respectively. For BT549 sh742- and sh528-transduced tumor cells, 60% and40% decreases in invasion through Matrigel™ were observed. Suppressionof QSOX1 expression in BT474 cells leads to an 85% reduction in invasionof both sh742- and sh528-transduced tumor cells. These data suggest thatQSOX1 plays a role in regulating invasive behavior in vitro irrespectiveof breast tumor subtype and hormone receptor status.

To prove that suppression of QSOX1 protein expression was responsiblefor loss of tumor cell invasion, we performed a rescue experiment inwhich recombinant human QSOX1 (rhQSOX1, generously provided by Dr. ColinThorpe) was added to shQSOX1-MCF7, shQSOX1-BT549 and shQSOX1-BT474 cellsduring the invasion assay. As a control for the enzymatically activeQSOX1, a mutant rhQSOX1 in which the CxxC motif in the thioredoxin-1domain was mutated to AxxA (rhQSOX1(AA), generously provided by Dr.Debbie Fass) was added to the invasion assay. Addition of enzymaticallyactive rhQSOX1 rescued the invasive phenotype of the shQSOX1-transducedtumor cells (FIG. 10d-f ), while the addition of the rhQSOX1(AA) did notrescue invasion of the shQSOX1-transduced tumor cells.

Decrease in QSOX1 Leads to a Decrease in Matrix MetalloproteinaseActivity

Since knockdown of QSOX1 resulted in decreased breast tumor cellinvasion, it was important to determine a mechanism for how QSOX1 mightfacilitate invasion. Matrix metalloproteinases (MMP) have been shown toplay key roles in breast tumor invasion and metastasis [21]. Both MMP-2and -9 mRNA and protein levels have been shown to contribute to breasttumor invasion, metastasis and angiogenesis [22]. Since previous workdemonstrated that QSOX1-S is secreted into the extracellular matrixwhere MMPs are activated, we hypothesized that QSOX1 might help activateMMP-2 and -9 proteins. MCF7 and BT549 cells transduced with shScramble,sh742 and sh528 were plated at equal densities and allowed to incubatein serum free media for 48 hours, after which the supernatants werecollected and analyzed by gelatin zymography to determine if the loss ofQSOX1 leads to a decrease in the functional activity of MMP-2 and -9.

Initial analysis of the results indicates that MCF7 and BT549 possesssimilar MMP profiles even though it is known that BT549 cells are moreinvasive. Luminal B-like breast tumor cell lines BT474 and ZR75 expressdo not secrete detectable levels of MMPs [23-25]. However, both MCF7 andBT549 supernatants contain MMP-9 homodimer (130 kDa), a large amount ofproteolytically active pro-MMP-9 (92 kDa) with lesser concentrations ofproteolytically active pro-MMP-2 (72 kDa).

We found that supernatants from MCF7 cells transduced with sh742 andsh528 showed a 70% and 77% decrease, respectively, in pro-MMP9 activitycompared to shScramble (FIG. 11a ). MCF7 supernatants from cellstransduced with sh742 and sh528 also showed a 50% and 60% decrease inactive MMP-9 (a-MMP-9) as well (FIG. 11a ). Supernatants from BT549cells transduced with sh742 and sh528 showed a 34% and 88% decrease,respectively, in MMP-9 (FIG. 11b ). Decreases in the proteolyticactivity of MMP-9, using gelatin as a substrate, provide a mechanism forthe shQSOX1-mediated suppression of invasion through Matrigel.

To extend our hypothesis that QSOX1 is activating or modifying MMPspost-translationally, we performed a western blot on total cell lysatefrom MCF7 and BT549 transduced cells as well as performed quantitativereal time PCR (qPCR) to determine if the loss of QSOX1 affected MMPprotein and RNA levels (FIG. 11c,d ). Our results indicate that theintracellular amount of MMP-2 and -9 protein is similar between theuntreated, shScramble, sh742 and sh528 samples in MCF7 and BT549 cells(FIG. 11c ). FIG. 11d demonstrates that the loss of QSOX1 also has nosignificant effect on the transcriptional activity of MMP-2 and -9.These results add confidence to our hypothesis that QSOX1 is involved inthe post-translational activation of MMPs.

Discussion for Example 4

To determine if QSOX1 was over-expressed in breast cancer, a GOBOanalysis was performed using data from over 1,800 breast cancer cases. Aprominent finding in this analysis is that the highest levels of QSOX1expression in Luminal B breast cancer correlate with very poor RFS andOS (FIG. 12c, d ). The median survival in patients with Luminal B breastcancer who over-express QSOX1 is approximately four years. Theprognostic power of QSOX1 expression for RFS and OS increases whenLuminal B breast cancer cases are divided into quintiles using the GOBOanalysis tool for which patients with the highest fifth expression ofQSOX1 have RFS of less than two years and OS of less than three years.In support of our GOBO analysis, showing that expression of QSOX1 is anindicator of poor OS and RFS in Luminal B breast cancer patients, weperformed IHC on breast TMA samples. We were able to confirm thatexpression of QSOX1 significantly correlates with ER+ breast tumor(P=0.0013) cells as well as correlating with high Ki-67 expression(P=0.0011), further supporting a role for QSOX1 in cellularproliferation (FIG. 13a ). Additionally, over-expression of QSOX1 mRNAin the GOBO analysis and high levels of protein in IHC correlate withincreasing tumor grade in our breast tumor TMA analyses (FIG. 13; Table1). Expression of QSOX1 did not correlate with survival in HER2 enrichedtumors, ER− tumors or in tumors subtyped as basal-like. Importantly, inpatients who did not receive systemic therapy (presumably due todiagnosis of very early stage disease), QSOX1 appears to be a predictorof poor OS. However, this association was not strong until more thanfive years post diagnosis.

Tumor cells in which QSOX1 expression was suppressed using shRNAs grewat less than half the rate of shScramble and untreated controls in MCF7,BT549 and BT474 cells (FIG. 14e-j ). The results of the MTT and TrypanBlue assays confirm our breast TMA findings showing that high expressionof QSOX1 correlates with high Ki-67 expression. Our attempt to explainthe decrease in cell growth by abnormal cell cycle regulation, apoptosisand autophagy suggests that QSOX1 is not involved in apoptosis, orautophagy, but may marginally affect cell cycle, as we observed a stallin G₁ and an increase in S phase in MCF7 cells (luminal-like) and aninsignificant increase in G₁ and a decrease in S phase in BT474(luminal-like) cells compared to shScramble controls. However, therewere no observable changes in BT549 cells (basal-like). These results,combined with our findings in PDA suggest that QSOX1 is unlikely to playa significant role in cell cycle. Our analysis of apoptosis andautophagy as a second possible mechanism contributing to the observeddecrease in cell growth did not reveal significant increases in AnnexinV/PI or LC3 expression (autophagy) in our shRNA treated cells. We alsodid not observe any increases in Trypan Blue positive cells during ourcell growth assays compared to our shScramble control (data not shown).

The ability of a tumor cell to invade is one of several hallmarks ofcancer [27]. Based on our results showing that QSOX1 over-expression inpancreas tumor cells contributes to invasion, we hypothesized that theover-expression of QSOX1 in breast adenocarcinoma would elicit a similarphenotype. MCF7, BT549 and BT474 cells transduced with QSOX1 shRNAsexhibited significant decreases in their ability to degrade basementmembrane components and invade through Matrigel™ (FIG. 10a-c ). MCF7cells are a poorly invasive, Luminal A-like breast cancer cell line,while BT549 (basal-like) and BT474 (Luminal B-like) cells are highlyinvasive [28, 29]. Although the invasive capabilities are dramaticallydifferent between these cell lines, QSOX1 knock-down suppressed growthand invasion in all cell lines irrespective of the level of QSOX1expression (FIG. 13a ) and molecular tumor subtype. Addition ofexogenous recombinant QSOX1 protein to shQSOX1 transduced tumor cellsrescued their invasive properties (FIG. 10d-f ), confirming dataindicating that QSOX1 is secreted into the extracellular matrix.

These findings indicate the advantage that QSOX1 provides to breast andpancreas tumors may be highly conserved and universal among other tumortypes. What we can conclude from our human TMA analysis of QSOX1 proteinexpression is that QSOX1 is a very specific marker of tumor cells andthat the expression of QSOX1 correlates with increased proliferation(high Ki-67) and an increase in tumor grade consistent with thecharacteristics of highly invasive tumors.

MMPs are a family of proteases that are involved in the degradation ofbasement membrane components contributing to tumor cell invasion andproliferation [34]. In breast tumors, gelatinases, MMP-2 and MMP-9 havebeen shown to play a significant role in growth and metastasis, as theirexpression is correlated with aggressive forms of breast cancer [25, 34,35]. Gelatinases are secreted into the extracellular matrix in theirinactive, pro-form where they can be activated through either a cysteineswitch or shift in the prodomain mediated by integrins and laminin inbasement membranes and structural proteins, such as vimentin [25]. Thiolbinding proteins, such as glutathione, have also been shown to help foldand activate MMPs [35]. Our data indicate that MMPs could be onesubstrate of QSOX1. To address this we performed gelatin zymography toassess functional activity MMPs. Our data reveal that knockdown of QSOX1protein expression in both MCF7 and BT549 cells leads to a decrease inMMP-9 functional activity compared to shScramble control (FIG. 11a, b ).While the functional activity of MMP-2 and -9 was suppressed, mRNAencoding MMP-2 and -9 remained relatively constant in MCF-7 cells andincreased in BT549 cells (FIG. 11c, d ). BT474 cells unfortunately donot express or secrete levels of MMP-2 and -9 detectable by gelatinzymography [25, 36]. Interestingly, when we knock down QSOX1 in BT474cells we observe the same phenotypic effects indicating that there aremultiple substrates of QSOX1 contributing to our observed decrease incellular proliferation and invasion. Taken together, the data suggestthat QSOX1 may post-translationally activate MMPs.

QSOX1 is expressed during embryonic development in mouse and rat duringkey migratory stages [37]. This developmental data combined with ourresults indicating that QSOX1 expression facilitates degradation ofbasement membranes suggests that tumor cells over-express QSOX1 to allowthem to break down basement membranes and invade into adjacent tissuesor into circulation. QSOX1 expression in Luminal B subtype can helpfurther stratify which tumors are likely to be more aggressive, leadingto poor overall survival. Notably from these data we can project thattargeting QSOX1 irrespective of tumor subtype could help to slow tumorcell proliferation as well as tumor cell invasion. This finding providesanother tool for physicians and their patients to decide whether to moreaggressively treat patients with Luminal B breast cancer whose tumorsexpress high levels of QSOX1.

Material and Methods for Example 4 Cell Culture

Breast adenocarcinoma MCF7, MDA-MB-468, MDA-MB-453, BT474, ZR75, BT549and MDA-MB-231 cancer cell lines were cultured in DMEM with 10% fetalbovine serum (FBS) (Gibco). Immortal human non-tumorigenic breastepithelial cells (MCF10A) were cultured in Clontech KGM-2 karotinocytemedia (Gibco). All cell lines were grown at 37° C. with 5% CO₂. All celllines tested negative for mycoplasma contamination using, Venor GeMMycoplasma Detection Kit, (Sigma).

Immunohistochemistry (IHC) and Scoring of Staining Intensity

Breast tumor microarray slides were generated from 153 different breastcancer patients. Each patient's tumor was represented in triplicate onthe slides. Immunohistochemistry on breast tumor tissue microarraysamples was performed exactly as previously described [16]. Afterstaining the TMA slides with anti-QSOX1 rabbit polyclonal antibody, aboard certified pathologist (ITO) scored the staining pattern as i) thepercentage of cells with IHC staining for QSOX1 protein expression inthe core tumor tissue sample (0: no staining, 1 (Low): 1 to 33%, 2(Intermediate): 34 to 66%, 3 (High): 67 to 100%), and ii) the intensityof the antibody stain (0: no staining, 1: weak, 2: moderate, 3: strongstaining intensity).

All samples were pre-existing and de-identified and, therefore, exemptfrom review by the human subjects Institutional Review Board at ArizonaState University.

Statistical Assessment of QSOX1 IHC with Molecular Subtypes of BreastCancer

There were 153 patient tissue samples in triplicate stained withanti-QSOX1 rabbit polyclonal Ab (Proteintech, Chicago, Ill., USA). IHCstaining was scored by a board certified pathologist (ITO). The amountand intensity of QSOX1 staining/expression was scored on a scale of 0 to3. The first IHC score number represents the percentage of cellsstaining (0: No staining, 1: 1 to 33%, 2: 34 to 66%, 3:67 to 100%), andthe second represents intensity (0: No staining, 1: weak, 2: moderate,3: strong staining intensity). We grouped the scores into fourcategories: 0 (No staining), 11/12/21 (Low staining), 22/13/31(Intermediate staining) and 32/33/23 (High staining).

To evaluate the relationship between markers (Tumor grade, Her2, CK5/6,and Ki-67) and QSOX1, Pearson's chi-square test was performed. Usingtwo-sided P-values, statistical significance will be set at P≦0.05.

Generation of Short Hairpin (Sh) RNA and Lentiviruses Production

As described in previous examples.

Generation of shQSOX1-Transduced Tumor Cell Lines

Stable transduction of sh742, sh528, and shScramble into MCF7, BT474 andBT549 cell lines was performed by first seeding the cells at 6×10⁵cells/well in a 6 well plate and incubating overnight. The next day thecells were transduced by adding 8 ug/mL polybrene (Millipore) and 200 ulsh742, sh528, and shScramble lentivirus produced from 293T cells to eachwell. The cells were then incubated for 24 hours. The following dayfresh DMEM with 10% FBS was added, containing 1 ug/mL puromycin (Sigma)to select for the transduced cells. QSOX1 knockdown was measured bywestern blot.

SDS-PAGE-Western Blotting

Western blotting was performed using cell lysates from MCF1OA, MCF7,MDA-MB-468, MDA-MB-453, BT474, ZR 75, BT549 and MDA-MB-231. Cell lysateswere generated by harvesting 2.5×10⁶ cells by centrifugation followed bylysis using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA,and 1% Triton X-100) with 1× SigmaFAST Protease Inhibitor CocktailTablet, EDTA Free. Protein in the cell lysate was measured using themicro BCA protein assay kit (Thermo Scientific). All samples were thennormalized to 2 mg/mL (20 ug total protein per lane). Samples were runon 10% SDS-polyacrylamide gels then transferred onto Immun-Blot™ PVDFMembranes (Bio-Rad). Rabbit polyclonal anti-QSOX1 (ProteinTech), rabbitpolyclonal anti-alpha-tubulin (Cell Signaling), rabbit polyclonalanti-MMP-2 and -9 (Sigma), mouse monoclonal caspase 3 (Cell Signalling),and rabbit polyclonal LC3 (Cell Signalling) antibodies were dilutedaccording to the manufacturers' instructions and as determined inpreliminary experiments, in 1% BSA in 1×TBS+0.01% Tween-20 and incubatedovernight. Goat anti-rabbit or anti-mouse IgG-alkaline phospatase or HRPsecondary antibody was used at a 1:5000 dilution and incubated with theblot for 1 h followed by washing. BCIP/NBT substrate (Pierce Chemical,Rockford, Ill.) was added and the blot was developed at room temperature(RT) for approximately 10 minutes for alkaline phosphatase secondaryantibody. For samples incubated in goat anti-rabbit or mouse HRPsecondary the blots were developed using Novex ECL ChemiluminescentSubstrate Reagent Kit. Quantification of band intensity was measuredusing Image J and is presented as percent change from the scrambledshRNA control. All gel images were annotated and processed usingPhotoshop CS3.

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay

Cells were seeded at 3×10³ cell/well in a 96-well plate in triplicate,and incubated at 37° C., 5% CO₂ over the course of 5 days. The MTT assaywas performed over a 5 day period according to the manufacturer'sinstructions (Invitrogen-Molecular Probes, Vybrant MTT CellProliferation Assay Kit). Results are presented as mean+/−S.D. Student'stwo-tailed t-Test was performed to determine significance.

Trypan Blue Live/Dead Cell Growth Assay

Cells were seeded at 2.5×10⁴ cells/well in a 12-well plate intriplicate, and incubated at 37° C., 5% CO₂ over the course of 5 days.The cells were removed with Cell Stripper, pelleted and brought back upin 1mL PBS. A 30 ul aliquot was then used to determine total cellnumber. The cells were stained at a 1:1 ratio with 0.1% Trypan Blue andare reported as total number of live cells.

RNA Isolation and cDNA Synthesis

Total RNA isolation was performed according to the manufacturer'sinstructions for animal cells using spin technology (RNeasy Mini Kit,Qiagen). After RNA was isolated from each sample was reverse transcribedwith qScript cDNA Sythesis Kit, Quanta Biosciences according to themanufacturer's instructions.

Quantitative Real Time PCR (qPCR)

The relative level of GAPDH, QSOX1-L, QSOX1-S, MMP-2 and MMP-9 wereanalyzed in each sample by qPCR. Each cDNA sample was normalized to 100ng/μl in molecular grade water along with 100 nM final concentration ofeach primer and 1× final concentration of PerfeCta SYBR Green Fast Mix(Quanta Biosciences, Gaithersburg, Md., USA). ROX to a final volume of10 ul. qPCR was performed using PerfeCTa SYBR Green FastMix, ROX fromQuanta Biosciences (Quanta Biosciences, Gaithersburg, Md., USA) on anABI7900HT thermocycler, Applied Biosystems Inc. (Life Technologies,Grand Island, N.Y., USA) Reaction protocol: initial denaturation was asfollows—95° C. for 3 minutes; PCR Cycling (40 cycles) 1.) 95° C., 30sec. 2.) 55° C., 30 sec. 3.) 72° C., 1 minute; Melt Curve (Dissociationstage). The primer sequences for the genes analyzed are:

GAPDH Forward (SEQ ID NO: 30) 5′-GGCCTCCAAGGAGTAAGACC; GAPDH Reverse(SEQ ID NO: 31) 5′-AGGGGTCTACATGGCAACTG; QSOX1-S Forward (SEQ ID NO: 32)5′-TGGTCTAGCCACAACAGGGTCAAT; QSOX1-S Reverse (SEQ ID NO: 33)5′-TGTGGCAGGCAGAACAAAGTTCAC; QSOX1-L Forward (SEQ ID NO: 34)5′-TTGCTCCTT GTCTGGCCTAGAAGT; QSOX1-L Reverse (SEQ ID NO: 35)5′-TGTGTCAAAGGAGCTCTCTCTGTCCT; MMP-2 Forward (SEQ ID NO: 36)5′-TTGACGGTAAGGACGGACTC; MMP-2 Reverse (SEQ ID NO: 37)5′-ACTTGCAGTACTCCCCATCG; MMP-9 Forward (SEQ ID NO: 38)5′-TTGACAGCGACAAGAAGTGG; and MMP-9 Reverse (SEQ ID NO: 39)5′-CCCTCAGTGAAGCGGTACAT..Each reaction was performed in triplicate with the data representing theaverages of one experiment.

In the shRNA experiment, expression of MMPs was normalized to thenon-targeted GAPDH to determine ΔCq. ΔCq replicates were thenexponentially transformed to the ΔCq expression after which they wereaveraged±standard deviation. The average was then normalized to theexpression of the shScramble control to obtain the ΔΔCq expression.Significance was determined using the Student's two-tailed t-Test.

Boyden Chamber and Invasion Recovery Assay

Invasion assays were performed using BD BioCoat™ BD Matrigel™ andnon-Matrigel™ control Invasion chambers with 8.0 μm pore sizepolyethylene terephthalate (PET) membrane inserts in 24-well format. Theassay was performed according to the manufacturer's instructions (BDBioscience). 4×10⁴ cells/well were seeded into the inner Matrigel™chamber in serum free DMEM. The outer chamber contained 10% FBS in DMEM.MCF7, BT474 and BT549 cells were incubated for 72, 48 and 48 hours,respectively at 37° C., 5% CO₂. For invasion rescue assays MCF7, BT474and BT549 cells were incubated with 50 nM rQSOX1 as well ascatalytically inactive mutant rQSOX1 (rQSOX1-AA). Cells that invadedthrough the Matrigel™ and migrated through the pores onto the bottom ofthe insert were fixed in 100% methanol and then stained in hematoxylin(Invitrogen). The total number of invading cells was determined bycounting the cells on the underside of the insert from triplicate wells(6 fields per insert) at 20× magnification. The extent of invasion wasexpressed as the average±standard deviation. Significance was determinedusing the Student's two-tailed t-Test. Results presented are from one ofthree independent experiments.

Gelatin Zymography

The identification of MMP was performed using gelatin zymography.Zymography experiments were performed essentially as previouslydescribed by Katchman et al. Minor changes in the protocol are theinclusion of untreated MCF7 and BT549 cells as well as shorthairpin-transduced cells were seeded at 5×10⁵ cells/well (12 wellplates) in DMEM with 10% FBS. The next day, cells were then washed with1×PBS and the media was changed to serum-free DMEM and incubated for 48hours instead of 24 hours before being collected and proteinconcentrations determined using a BCA assay. Quantification of bandintensity was measured using Image J and is presented as percent changefrom the scrambled shRNA control.

REFERENCE FOR EXAMPLE 4

-   1. Sgroi D C: Preinvasive breast cancer. Annu Rev Pathol 2010,    5:193-221.-   2. Cancer Facts and Figures. American Cancer Society (ACS); 2012.    See the ACS web site    cancer.org/research/cancerfactsfigures/cancerfactsfigures/cancer-facts-figures-2012-   3. Siegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA    Cancer J Clin 2012, 62:10-29.-   4. Antwi K, Hostetter G, Demeure M, Decker G, Ruiz Y, Sielaff T,    Koep L, Lake D: Analysis of human plasma peptidome reveals potential    biomarker for pancreatic cancer. J Proteome Res 2009, 8:4722-4731.-   5. Katchman B A, Antwi K, Hostetter G, Demeure M J, Watanabe A,    Decker G A, Miller L J, Von Hoff D D, Lake D F: Quiescin sulfhydryl    oxidase 1 promotes invasion of pancreatic tumor cells mediated by    matrix metalloproteinases. Mol Cancer Res 2011, 9:1621-1631.-   6. Alon A, Heckler E J, Thorpe C, Fass D: QSOX contains a    pseudo-dimer of functional and degenerate sulfhydryl oxidase    domains. FEBS Lett 2010, 584:1521-1525.-   7. Coppock D L, Thorpe C: Multidomain flavin-dependent sulfhydryl    oxidases. Antioxid Redox Signal 2006, 8:300-311.-   8. Heckler E J, Alon A, Fass D, Thorpe C: Human quiescin-sulfhydryl    oxidase, QSOX1: probing internal redox steps by mutagenesis.    Biochemistry 2008, 47:4955-4963.-   9. Chakravarthi S, Jessop C E, Willer M, Stirling C J, Bulleid N J:    Intracellular catalysis of disulfide bond formation by the human    sulfhydryl oxidase, QSOX1. Biochem J 2007, 404:403-411.-   10. Mairet-Coello G, Tury A, Esnard-Feve A, Fellmann D, Risold P Y,    Griffond B: FAD-linked sulfhydryl oxidase QSOX: topographic,    cellular, and subcellular immunolocalization in adult rat central    nervous system. J Comp Neurol 2004, 473:334-363.-   11. Tury A, Mairet-Coello G, Poncet F, Jacquemard C, Risold P Y,    Fellmann D, Griffond B: QSOX sulfhydryl oxidase in rat    adenohypophysis: localization and regulation by estrogens. J    Endocrinol 2004, 183:353-363.-   12. Coppock D L, Cina-Poppe D, Gilleran S: The quiescin Q6 gene    (QSCN6) is a fusion of two ancient gene families: thioredoxin and    ERV1. Genomics 1998, 54:460-468.-   13. Alon A, Grossman I, Gat Y, Kodali V K, DiMaio F, Mehlman T,    Haran G, Baker D, Thorpe C, Fass D: The dynamic disulphide relay of    quiescin sulphydryl oxidase. Nature 2012, 488:414-418.-   14. Thorpe C, Hoober K L, Raje S, Glynn N M, Burnside J, Turi G K,    Coppock D L: Sulfhydryl oxidases: emerging catalysts of protein    disulfide bond formation in eukaryotes. Arch Biochem Biophys 2002,    405:1-12.-   15. Song H, Zhang B, Watson M A, Humphrey P A, Lim H, Milbrandt J:    Loss of Nkx3.1 leads to the activation of discrete downstream target    genes during prostate tumorigenesis. Oncogene 2009, 28:3307-3319.-   16. Sorlie T, Perou C M, Tibshirani R, Aas T, Geisler S, Johnsen H,    Hastie T, Eisen M B, van de Rijn M, Jeffrey S S, Thorsen T, Quist H,    Matese J C, Brown P O, Botstein D, Lonning P E, Borresen-Dale A L:    Gene expression patterns of breast carcinomas distinguish tumor    subclasses with clinical implications. Proc Natl Acad Sci USA 2001,    98:10869-10874.-   17. Ringner M, Fredlund E, Hakkinen J, Borg A, Staaf J: GOBO: gene    expression-based outcome for breast cancer online. PLoS One 2011,    6:e17911.-   18. Perou C M, Sorlie T, Eisen M B, van de Rijn M, Jeffrey S S, Rees    C A, Pollack J R, Ross D T, Johnsen H, Akslen L A, Fluge O,    Pergamenschikov A, Williams C, Zhu S X, Lønning P E, Borresen-Dale A    L, Brown P O, Botstein D: Molecular portraits of human breast    tumours. Nature 2000, 406:747-752.-   19. Soule H D, Maloney T M, Wolman S R, Peterson W D Jr, Brenz R,    McGrath C M, Russo J, Pauley R J, Jones R F, Brooks S C: Isolation    and characterization of a spontaneously immortalized human breast    epithelial cell line, MCF-10. Cancer Res 1990, 50:6075-6086.-   20. Tait L, Soule H D, Russo J: Ultrastructural and    immunocytochemical characterization of an immortalized human breast    epithelial cell line, MCF-10. Cancer Res 1990, 50:6087-6094.-   21. Morel C, Adami P, Musard J F, Duval D, Radom J, Jouvenot M:    Involvement of sulfhydryl oxidase QSOX1 in the protection of cells    against oxidative stress-induced apoptosis. Exp Cell Res 2007,    313:3971-3982.-   22. Plati J, Bucur O, Khosravi-Far R: Apoptotic cell signaling in    cancer progression and therapy. Integr Biol (Camb) 2011, 3:279-296.-   23. Klionsky D J: The molecular machinery of autophagy and its role    in physiology and disease. Semin Cell Dev Biol 2010, 21:663.-   24. Radisky E S, Radisky D C: Matrix metalloproteinase-induced    epithelial-mesenchymal transition in breast cancer. J Mammary Gland    Biol Neoplasia 2010, 15:201-212.-   25. Kohrrnann A, Kammerer U, Kapp M, Dietl J, Anacker J: Expression    of matrix metalloproteinases (MMPs) in primary human breast cancer    and breast cancer cell lines: New findings and review of the    literature. BMC cancer 2009, 9:188.-   26. Khoo B Y, Miswan N, Balaram P, Nadarajan K, Elstner E:    Modification of MCF-10A Cells with Pioglitazone and Serum-Rich    Growth Medium Increases Soluble Factors in the Conditioned Medium,    Likely Reducing BT-474 Cell Growth. Int J Mol Sci 2012,    13:5607-5627.-   27. Hanahan D, Weinberg R A: Hallmarks of cancer: the next    generation. Cell 2011, 144:646-674.-   28. Polyak K: Heterogeneity in breast cancer. J Clin Invest 2011,    121:3786-3788.-   29. Rizki A, Weaver V M, Lee S Y, Rozenberg G I, Chin K, Myers C A,    Bascom J L, Mott J D, Semeiks J R, Grate L R, Mian I S, Borowsky A    D, Jensen R A, Idowu M O, Chen F, Chen D J, Petersen O W, Gray J W,    Bissell M J: A human breast cell model of preinvasive to invasive    transition. Cancer Res 2008, 68:1378-1387.-   30. Hu M, Polyak K: Molecular characterisation of the tumour    microenvironment in breast cancer. Eur J Cancer 2008, 44:2760-2765.-   31. Michor F, Polyak K: The origins and implications of intratumor    heterogeneity. Cancer Prev Res (Phila) 2010, 3:1361-1364.-   32. Bacac M, Stamenkovic I: Metastatic cancer cell. Annu Rev Pathol    2008, 3:221-247.-   33. Martin K J, Patrick D R, Bissell M J, Fournier M V: Prognostic    breast cancer signature identified from 3D culture model accurately    predicts clinical outcome across independent datasets. PLoS One    2008, 3:e2994.-   34. Kessenbrock K, Plaks V, Werb Z: Matrix metalloproteinases:    regulators of the tumor microenvironment. Cell, 141:52-67.-   35. Bauvois B: New facets of matrix metalloproteinases MMP-2 and    MMP-9 as cell surface transducers: outside-in signaling and    relationship to tumor progression. Biochim Biophys Acta 2012,    1825:29-36.-   36. Jin Q, Yuan L X, Boulbes D, Baek J M, Wang Y N, Gomez-Cabello D,    Hawke D H, Yeung S C, Lee M H, Hortobagyi G N, Hung M C, Esteva F J:    Fatty acid synthase phosphorylation: a novel therapeutic target in    HER2-overexpressing breast cancer cells. Breast Cancer Res 2010,    12:R96.-   37. Portes K F, Ikegami C M, Getz J, Martins A P, de Noronha L,    Zischler L F, Klassen-   G, Camargo A A, Zanata S M, Bevilacqua E, Nakao L S: Tissue    distribution of quiescin Q6/sulfhydryl oxidase (QSOX) in developing    mouse. J Mol Histol 2008, 39:217-225.

What is claimed is:
 1. A method for tumor treatment, comprisingadministering to a subject having a tumor an amount effective of aninhibitor of quiescin sulfhydryl oxidase 1 (QSOX1) expression and/oractivity, or a pharmaceutically acceptable salt thereof, to treat thetumor.
 2. The method of claim 1, wherein the inhibitor of QSOX1 isselected from the group consisting of anti-QSOX1 antibodies,QSOX1-binding aptamers, QSOX1 antisense oligonucleotides, QSOX1 siRNA,and QSOX1.
 3. The method of claim 1, wherein the tumor is a tumor thatover-expresses QSOX1 compared to control.
 4. The method of claim 1,wherein the tumor is a pancreatic tumor
 5. The method of claim 4,wherein the pancreatic tumor comprises a pancreatic adenocarcinoma. 6.The method of claim 1, wherein the tumor is a breast tumor.
 7. Themethod of claim 6, wherein the tumor is an estrogen receptor positive(ER+) breast tumor.
 8. The method of claim 6, wherein the breast tumoris a Luminal B breast tumor.
 9. The method of claim 1, wherein theinhibitor comprises an isolated nucleic acid molecule selected from thegroup comprising antisense, siRNA, miRNA, and/or shRNA having a nucleicacid sequence perfectly complementary to at least 10 contiguousnucleotides of SEQ ID NO:1 or SEQ ID NO: 2, or an RNA equivalentthereof.
 10. The method of claim 1, wherein the method limits tumormetastasis.
 11. A method inhibiting tumor metastasis, comprisingadministering to a subject having a tumor an amount effective of aninhibitor of quiescin sulfhydryl oxidase 1 (QSOX1) expression and/oractivity, or a pharmaceutically acceptable salt thereof, to inhibitmetastasis of the tumor.
 12. The method of claim 11, wherein theinhibitor of QSOX1 is selected from the group consisting of anti -QSOX1antibodies, QSOX1-binding aptamers, QSOX1 antisense oligonucleotides,QSOX1 siRNA, and QSOX1 shRNA.
 13. The method of claim 11, wherein thetumor is a breast tumor.
 14. The method of claim 11, wherein the tumoris a cytokeratin 5/6 negative breast tumor, or a Luminal B breast tumor.15. The method of claim 11, wherein the tumor is a pancreatic tumor. 16.The method of claim 11, wherein the inhibitor comprises an isolatednucleic acid molecule selected from the group comprising antisense,siRNA, miRNA, and/or shRNA having a nucleic acid sequence perfectlycomplementary to at least 10 contiguous nucleotides of SEQ ID NO: 1 orSEQ ID NO: 2, or an RNA equivalent thereof.